Functional Role of Fibrillin5 in Acclimation to Photooxidative Stress

Functional Role of Fibrillin5 in Acclimation to Photooxidative Stress Abstract The functional role of a lipid-associated soluble protein, fibrillin5 (FBN5), was determined with the Arabidopsis thaliana homozygous fbn5-knockout mutant line (SALK_064597) that carries a T-DNA insertion within the FBN5 gene. The fbn5 mutant remained alive, displaying a slow growth and a severe dwarf phenotype. The mutant grown even under growth light conditions at 80 µmol m–2 s–1 showed a drastic decrease in electron transfer activities around PSII, with little change in electron transfer activities around PSI, a phenomenon which was exaggerated under high light stress. The accumulation of plastoquinone-9 (PQ-9) was suppressed in the mutant, and >90% of the PQ-9 pool was reduced under growth light conditions. Non-photochemical quenching (NPQ) in the mutant functioned less efficiently, resulting from little contribution by energy-dependent quenching (qE). The ultrastructure of thylakoids in the mutant revealed that their grana were unstacked and transformed into loose and disordered structures. Light-harvesting complex (LHC)-containing large photosystem complexes and photosystem core complexes in the mutant were less abundant than those in wild-type plants. These results suggest that the lack of FBN5 causes a decrease in PQ-9 and imbalance of the redox state of PQ-9, resulting in misconducting both short-term and long-term control of the input of light energy to photosynthetic reaction centers. Furthermore, in the fbn5 mutant, the expression of genes involved in jasmonic acid biosynthesis was suppressed to ≤10% of that in the wild type under both growth-light and high-light conditions, suggesting that FBN5 functions as a transmitter of 1O2 in the stroma. Introduction In plants, light is not only a source of energy but also a source of information about their environment. The photosynthetic electron transfer driven by light can interact with molecular oxygen, leading to the formation of reactive oxygen species (ROS), mainly O2·– in PSI and mainly 1O2 in PSII (Asada 1999, Apel and Hirt 2004, Fischer et al. 2013). Production of ROS is significantly enhanced under photooxidative stress conditions where plants absorb excessive light relative to the photosynthetic activity. ROS attack various types of biological molecules such as DNA and proteins to damage cells/tissues, besides functioning as crucial cellular signals. In the photosynthetic apparatus, the synthesis of the PSII reaction center protein D1 is inhibited by ROS to impair photosynthetic function (Nishiyama et al. 2001, Vass 2012). Such impairment of PSII tends to remove the balance between both photosystems, falling into a vicious circle leading to further production of ROS. On the other hand, there is growing evidence that ROS can also serve as a protection mechanism against photoinhibition to prevent further damage of the system (Fischer et al. 2013, Foyer et al. 2017). To manage the resulting production of ROS, chloroplasts contain a variety of antioxidant mechanisms including soluble and lipophilic low molecular weight antioxidants, detoxification enzymes and repair mechanisms (Tarrago et al. 2008, Tripathi et al. 2009, Pospíšil 2014). For example, the plastoquinone (PQ) pool located on the acceptor side of PSII has been attributed a role in the regulation of gene expression and enzyme activities through its redox state (Pfannschmidt et al. 2001, Tikkanen et al. 2012, Rochaix 2013, Petrillo et al. 2014), as well as acting as an antioxidant in plant leaves to play a central photoprotective role (Ksas et al. 2015). In chloroplasts and chromoplasts, there are tiny granules, so-called plastoglobules (PGs) which function as a reservoir to accumulate lipids such as tocopherols and carotenoids that are antioxidants to quench ROS. PGs are lipoprotein bodies surrounded by a monolayer of phospholipid that is contiguous with the stroma-side leaflet of the thylakoid membrane (Austin et al. 2006), and contain structural proteins called fibrillins (FBNs) and enzymatic proteins as well as antioxidants (Brehelin et al. 2007). PGs vary in size and number during plastid development and differentiation, and are more abundant in response to environmental stresses [e.g. high light (HL), drought, high salinity and exposure to ozone] and during senescence (Rey et al. 2000, Simkin et al. 2007, Singh et al. 2010, Youssef et al. 2010, Ariizumi et al. 2014, Martinis et al. 2014). FBNs, which were first discovered in fibrils, the suborganellar structures in chromoplasts, are generally associated with PGs in leaf tissue and also termed plastid lipid-associated proteins (PAPs) or plastoglobulins (Pozueta-Romero et al. 1997, Kessler et al. 1999). FBNs are a large protein family present in photosynthetic organisms ranging from cyanobacteria to higher plants, and can be divided to 12 phylogenetic groups in which Groups 1, 3 and 7 have two ortholog proteins each (Singh and McNellis 2011). All FBNs possess chloroplast transit peptide sequences in their N-terminus. van Wijk’s group demonstrated, with proteome analyses of PGs and thylakoid membranes from Arabidopsis thaliana (Arabidopsis), that at least eight FBNs (FBN1a/1b/2/3a/4/7a/7b/8) are bound to PGs (Friso et al. 2004, Ytterberg et al. 2006). They further clarified the localization of FBNs in chloroplasts by mass spectrometry of isolated PGs and quantitative comparison with the proteomes of unfractionated leaves, thylakoids and stroma (Lundquist et al. 2012): the PG/thylakoid and PG/stroma abundance ratios of FBN1a were 34 and 176, respectively, which confirms that almost all of FBN1a is localized on PGs. From this study, FBN1a/1b/2/4/7a/7b/8 were localized in PGs and constituted 53% of the PG proteome mass, while FBN3a/3b/6/9 were localized in thylakoid membranes. FBN10 was present on both PGs and thylakoid membranes. In recent years, experimental research has made it clear that some FBNs, known as major structural proteins of PGs, are involved in the adaption to biotic/abiotic environmental stresses. FBN1a/1b/2-knockdown plants increased their sensitivity to photoinhibition of PSII, resulting in impairment of long-term acclimation to photooxidative stress imposed by HL combined with cold. Jasmonate biosynthesis related to light/cold stress is also regulated by the accumulation of PG-associated FBN1/2 proteins (Youssef et al. 2010). Gamez-Arjona et al. (2014) reported that FBN1a can interact with starch synthase 4 located in specific areas of the thylakoid membranes, suggesting that FBN1a functions in the regulation of synthesis of starch granules at specific regions of chloroplasts. FBN4 was also reported to be involved in the acquisition of resistance to the various stresses such as pathogenic infection and ozone exposure (Singh et al. 2010). FBN4-knockdown apple trees were more susceptible than their wild-type counterparts to the bacterial pathogen Erwinia amylovora and were more sensitive to ozone-induced tissue damage. PG osmiophilicity, which is considered to reflect the content of major antioxidants such as PQ, carotenoids and triacylglycerides, was decreased in FBN4-knockdown apple tree chloroplasts compared with the wild type. Indeed, the PQ level in mutant PGs was <10% of that in wild-type PGs (Singh et al. 2012). FBN4 is suggested to play a role in accumulating antioxidant lipids into PG, resulting in an increase in stress resistance. Little is known about the function and localization of FBN5 in stressed plants, compared with FBNs of major PG proteins. FBN5 belongs to Group 5, and is a unique minor protein of FBN family proteins because FBN5 is unlikely to be localized in PGs and thylakoid membranes, showing a low pI and the lowest hydrophobicity indices of the 16 FBN protein products (Lundquist et al. 2012). In our previous study, we initially identified FBN5 as one of the candidate genes functioning in freezing tolerance, using the activation-tagging callus lines generated by random insertion of strong enhancers (Otsubo et al. 2007). Recently, Kim et al. (2015) have demonstrated that FBN5 functions as a structural component involved in the biosynthesis of plastoquinone-9 (PQ-9). FBN5 binding to the hydrophobic solanesyl moiety, which is generated by solanesyl diphosphate (SPP) synthases (SPSs) 1 and 2, in the FBN5-B/SPS homodimeric complex stimulates their enzyme activities. They propose a model for PQ-9 biosynthesis in Arabidopsis whereby SPSs of the FBN5/dimeric SPS complex catalyze the sequential condensation of five molecules of isopentenyl diphosphate (IPP) with geranylgeranyl diphosphate (GGPP) to yield the all-trans form of SPP and facilitate both the conversion of SPP to PQ-9 and its translocation to the thylakoids and PGs. In this study, in order to clarify further an involvement of FBN5 in acclimation against abiotic environmental stresses, we have studied the structural and functional properties of FBN5 with fbn5-knockout and fbn5-complemented mutants. Results The T3 plants of the activation-tagged mutant containing the Cauliflower mosaic virus (CaMV) 35S enhancers, designated as the 18-16 line (Otsubo et al. 2007), were evaluated by freezing treatment at −14°C for 3 h (Fig. 1A). The calli from the 18-16 mutant line were more freezing tolerant than those from the wild-type plants. In this line, a single T-DNA activation-tag was inserted at position 3,058,129 on chromosome 5, by TAIL-PCR (thermal asymmetrical interlaced PCR) and Southern blotting (Fig. 1B). In four genes located in the vicinity of the enhancer insertion site, an At5g09820 gene, known as FBN5, was strongly expressed in the mutant line (Fig. 1C, D). Interestingly, there were two different sizes of transcripts of At5g09820 in reverse transcription–PCR (RT–PCR), of which the longer and shorter transcripts were tentatively named At5g09820a and At5g09820b, respectively. Fig. 1 View largeDownload slide Characterization of the activation-tagged line (18-16). (A) Evaluation of freezing tolerance with wild-type (WT) (left) and 18-16 (right) calli. After freezing at −14°C for 3 h, the calli were transferred to shoot-inducing medium (SIM). (B) Diagram showing the position of the T-DNA insertion (pPCVICEn4HPT) in the 18-16 line. HPT, hygromycin phosphotransferase; LB, left border; RB, right border; 4×En denotes four copies of 35S enhancer. (C, D) Expression profiles of genes located in the vicinity of the T-DNA insertion site in wild-type and 18-16 seedlings which were kept at 23 or 2°C for 5 d, analyzed by semi-quantitative RT–PCR using F2/R4 primers (C) and quantitative RT–PCR using F2/R3 or F2/R2 primers (D), respectively. Fig. 1 View largeDownload slide Characterization of the activation-tagged line (18-16). (A) Evaluation of freezing tolerance with wild-type (WT) (left) and 18-16 (right) calli. After freezing at −14°C for 3 h, the calli were transferred to shoot-inducing medium (SIM). (B) Diagram showing the position of the T-DNA insertion (pPCVICEn4HPT) in the 18-16 line. HPT, hygromycin phosphotransferase; LB, left border; RB, right border; 4×En denotes four copies of 35S enhancer. (C, D) Expression profiles of genes located in the vicinity of the T-DNA insertion site in wild-type and 18-16 seedlings which were kept at 23 or 2°C for 5 d, analyzed by semi-quantitative RT–PCR using F2/R4 primers (C) and quantitative RT–PCR using F2/R3 or F2/R2 primers (D), respectively. Cloning cDNAs of these transcripts, we found that they are two types of At5g08920 splicing variants, as shown in Fig. 2A: the transcript At5g09820a encodes 259 amino acids because of the existence of a stop codon in intron 5, while the transcript At5g09820b encodes the 273 amino acids of the full-length FBN5. Thus, in this study, At5g09820a and At5g09820b were conveniently named FBN5-259 and FBN5(-273), respectively. These FBN5 mRNA splicing variants were also reported by Kim et al. (2015). The expression of FBN5 under normal growth conditions was much greater than that of FBN5-259, while under cold conditions the latter became comparable with the former (Fig. 2B). To clarify the function of FBN5, we have investigated the Salk T-DNA insertion line (SALK_064597) in which FBN5 is functionally deleted. Two copies of T-DNA were inserted in tandem at position 3,057,168 of chromosome 5 in the first exon of FBN5, resulting in impairment of FBN5 transcription (Fig. 2C;Supplementary Fig. S1). A homozygous mutant of SALK_064597, which we named the fbn5 mutant, has been reported to be seedling lethal (Savage et al. 2013, Kim et al. 2015). We reconfirmed that continuous light conditions at 80 µmol m–2 s–1 caused all fbn5 plants to wilt and die. However, when the fbn5 plants were transferred to a 12 h:12 h day/night photoperiod at 80 µmol m–2s–1, approximately 20% of the plants did not die but were able to produce seeds 11–12 weeks after germination; they showed a dwarf phenotype (Supplementary Fig. S2). The fbn5 mutant was genotyped by PCR analysis using primers specific for the T-DNA and FBN5 (Supplementary Fig. S1; Table S1). A 550 bp band was detected with the F1/RBa2 but not with the F1/R1 primers, confirming that the mutant employed for this study is homozygous for the T-DNA insertion. The linkages of identical T-DNAs were reported to be frequently integrated at the same locus, independent of the transformation method or species used (De Neve et al. 1997). We have developed transgenic Arabidopsis lines, designated as fbn5/FBN5 and fbn5/FBN-259, overexpressing FBN5 and FBN5-259 cDNAs, respectively, driven under the control of the CaMV 35S promoter. The fbn5/FBN5 and fbn5/FBN5-259 lines exhibited mRNA levels increased by approximately 6- and 10-fold, respectively, compared with wild-type plants (Fig. 2C). Fig. 2 View largeDownload slide Alternative splicing of a FBN5 gene, and the organ-specific expression of splicing variants in wild-type plants. (A) Structures of FBN5 genomic DNA and two splicing variants. Roman numerals denote intron numbers. (B) Relative expression levels of FBN5-259 (white bar) and FBN5 (black bar). The tissues shown here were obtained from non-acclimated (left) and cold-acclimated (right) wild-type plants. The expression level of FBN5-259 in non-acclimated young leaves was set to 1. (C) Relative expression levels of FBN5 and FBN5-259 in wild-type, fbn5 (SALK_064597) and fbn5-complemented (fbn5/FBN5-259 and fbn5/FBN5) plants. Data are presented as the means ± SE (n = 4). Real-time RT–PCR primers for FBN5 (F2/R2) and FBN5-259 (F2/R3) were indicated in (A) and Supplementary Table S1. Fig. 2 View largeDownload slide Alternative splicing of a FBN5 gene, and the organ-specific expression of splicing variants in wild-type plants. (A) Structures of FBN5 genomic DNA and two splicing variants. Roman numerals denote intron numbers. (B) Relative expression levels of FBN5-259 (white bar) and FBN5 (black bar). The tissues shown here were obtained from non-acclimated (left) and cold-acclimated (right) wild-type plants. The expression level of FBN5-259 in non-acclimated young leaves was set to 1. (C) Relative expression levels of FBN5 and FBN5-259 in wild-type, fbn5 (SALK_064597) and fbn5-complemented (fbn5/FBN5-259 and fbn5/FBN5) plants. Data are presented as the means ± SE (n = 4). Real-time RT–PCR primers for FBN5 (F2/R2) and FBN5-259 (F2/R3) were indicated in (A) and Supplementary Table S1. Since the fbn5 plants transformed with cDNAs of FBN5 and FBN5-259 were phenotypically similar to wild-type and fbn5 plants, respectively, FBN5 was found to be a functional transcript (Supplementary Fig. S2). The structure of leaf tissues of 6-week-old wild-type, fbn5 and fbn5/FBN5 plants was examined with a light microscope (Fig. 3A–F). Compared with the wild-type plants, the fbn5 plants possessed cells with smaller size and thinner leaves, and fbn5/FBN5 plants exhibited thicker leaves. The mean number of chloroplasts per palisade mesophyll cell was 5.7 in fbn5 plants, while those in wild-type and fbn5/FBN5 plants were 10.1 and 9.5, respectively. Under the HL (800 µmol m-2 s-1) condition, starch accumulation was well observed in the chloroplast in wild-type and fbn5/FBN5 plants, but not in fbn5 plants (Fig. 3D–F). From TEM images shown in Fig. 3G–L, the fbn5 plants were found to possess abnormalities in chloroplast and thylakoid morphology, including swollen thylakoid vesicles in a granum, disorganized grana stacks, alterations in the relative proportions of grana and stroma thylakoids, and decreased starch granules. The chloroplast ultrastructure in the fbn5/FBN5 plants was similar to that of the wild-type plants, except for producing more abundant starch granules. Such architectural change in thylakoid membranes might affect the assembly of PSI/II reaction center complexes. Indeed, we found, with the native green gel analysis according to Allen and Staehelin (1991), that the amounts of light-harvesting complex (LHC)-containing large photosystem complexes (RC-LHC), photosystem complexes (RC-core) and LHCII trimers (LHCII-A, B, C and D) in fbn5 plants were much lower than those in wild-type and fbn5/FBN5 plants (Fig. 4). Since LHCII trimers are responsible for membrane stacking (Wan et al. 2014), a decrease in LHCII trimers may lead to the lack of grana stacking. Fig. 3 View largeDownload slide Light and ultramicroscopic photomicrographs of leaf tissue and chloroplast structures from wild-type, fbn5 and fbn5/FBN5 plants. (A–F) Cross-sections of rosette leaves of wild-type (A, D), fbn5 (B, E), and fbn5/FBN5 (C, F) plants. Upper and lower panels show the cross-section of leaves before and after 4 h HL treatment, respectively. Leaf pieces fixed and embedded for electron micrograph analysis were sliced 1 µm thick and stained with 0.5% toluidine blue. Scale bar = 50 μm. (G–L) Transmission electron micrograph of chloroplasts in wild-type (G, J), fbn5 (H, K) and fbn5/FBN5 (I, L) plants. Upper and lower panels show chloroplasts before and after 4 h HL treatment, respectively. Scale bar = 1 μm. Fig. 3 View largeDownload slide Light and ultramicroscopic photomicrographs of leaf tissue and chloroplast structures from wild-type, fbn5 and fbn5/FBN5 plants. (A–F) Cross-sections of rosette leaves of wild-type (A, D), fbn5 (B, E), and fbn5/FBN5 (C, F) plants. Upper and lower panels show the cross-section of leaves before and after 4 h HL treatment, respectively. Leaf pieces fixed and embedded for electron micrograph analysis were sliced 1 µm thick and stained with 0.5% toluidine blue. Scale bar = 50 μm. (G–L) Transmission electron micrograph of chloroplasts in wild-type (G, J), fbn5 (H, K) and fbn5/FBN5 (I, L) plants. Upper and lower panels show chloroplasts before and after 4 h HL treatment, respectively. Scale bar = 1 μm. Fig. 4 View largeDownload slide Native green gel electrophoresis of Chl–protein complexes isolated from wild-type, fbn5 and fbn5/FBN5 plants. Thylakoid membranes were isolated from 6-week-old plants, which had been grown under 12 h light/12 h dark or continuous light conditions for 2 weeks before the experiment. Separated bands are assigned according to Allen and Staehelin (1991) as follows: RC-LHC, large PSI and PSII complexes both with attached antenna; RC-Core, several partial PSI and PSII complexes that have been largely stripped of antennae; LHCII trimer, the trimeric form of the PSII antenna LHCII; SC, small complexes containing Chls. Fig. 4 View largeDownload slide Native green gel electrophoresis of Chl–protein complexes isolated from wild-type, fbn5 and fbn5/FBN5 plants. Thylakoid membranes were isolated from 6-week-old plants, which had been grown under 12 h light/12 h dark or continuous light conditions for 2 weeks before the experiment. Separated bands are assigned according to Allen and Staehelin (1991) as follows: RC-LHC, large PSI and PSII complexes both with attached antenna; RC-Core, several partial PSI and PSII complexes that have been largely stripped of antennae; LHCII trimer, the trimeric form of the PSII antenna LHCII; SC, small complexes containing Chls. The fbn5 plants were susceptible to long-day treatment such as a photoperiodic program of 16 h light:8 h dark or 24 h light (continuous light), and were withered. To evaluate how FBN5 affects photosynthetic capability, we have studied the effects of strong light on the maximum quantum efficiency of PSII photochemistry (Fv/Fm) in the wild-type and mutant plants (Fig. 5A). The HL treatment hardly decreased Fv/Fm values of the wild-type and fbn5/FBN5 plants during the time periods examined. On the other hand, Fv/Fm values of the fbn5 plants were decreased from the initial value of 0.6 to 0.02 by HL: longer exposure to HL rendered the fbn5 plants incapable of recovery of Fv/Fm under the subsequent growth light (GL, 80 µmol m–2 s–1) conditions (open symbols in Fig. 5A). To evaluate whether photodamage is enhanced and/or the repair is suppressed in fbn5 plants, we have studied the rates of photodamage to PSII by comparing photoinactivation kinetics in the presence of an inhibitor of protein synthesis, lincomycin, with the wild-type (Col-0) and fbn5 plants (Supplementary Fig. S3). Photoinactivation obeyed pseudo-first-order kinetics with respect to illumination time in both plants. The rate constant for photoinactivation, 0.073 h–1, at a light intensity of 80 µmol m–2 s–1 in fbn5 plants was much greater than that in the wild-type plants (0.0125 h–1). The addition of lincomycin increased rate constants to the same degree in both plants. These results have revealed that photodamage is enhanced in the fbn5 plants with little suppression of repair. We have also investigated the electron transfer activities around both photosystems, with thylakoid membranes isolated from wild-type and mutant leaves (Fig. 5B). Electron transfer activities around PSII in the fbn5 plants were drastically decreased by the HL exposure, while those for the wild-type and fbn5/FBN5 plants were not affected. Meanwhile, electron transfer activities of PSI remained intact after the HL treatment in all types of plants examined in this study. Taken together, our data provide evidence that dysfunction of FBN5 specifically induces photodamage of PSII. Fig. 5 View largeDownload slide Effects of HL on photosynthetic electron transfer in wild-type, fbn5 and fbn5/FBN5 plants. (A) The maximum quantum efficiency of PSII (Fv/Fm) under HL conditions. Filled circles, squares and triangles show the values for Fv/Fm of 6-week-old wild-type, fbn5/FBN5 and fbn5 plants, respectively, which were exposed to HL for a given time. Open symbols show the Fv/Fm values obtained with fbn5 plants which were transferred to GL conditions at a given time after HL exposure for 1, 3 or 6 h. Data are mean values of six separate experiments ± SD. (B) PSII/ PSI electron transfer activities before (gray) or after (white) 4 h HL treatment. Data are normalized to the values obtained with wild-type (WT) plants without any HL exposure. The rates of O2 evolution and consumption of 81 and 180 µmol O2 mg Chl–1 h–1, respectively, in wild-type plants were set at 100%. Data are presented as the means ± SD (n = 3–4). The asterisk marks statistically significant differences (P < 0.05 by Student’s t-test) relative to samples without any HL treatment. Fig. 5 View largeDownload slide Effects of HL on photosynthetic electron transfer in wild-type, fbn5 and fbn5/FBN5 plants. (A) The maximum quantum efficiency of PSII (Fv/Fm) under HL conditions. Filled circles, squares and triangles show the values for Fv/Fm of 6-week-old wild-type, fbn5/FBN5 and fbn5 plants, respectively, which were exposed to HL for a given time. Open symbols show the Fv/Fm values obtained with fbn5 plants which were transferred to GL conditions at a given time after HL exposure for 1, 3 or 6 h. Data are mean values of six separate experiments ± SD. (B) PSII/ PSI electron transfer activities before (gray) or after (white) 4 h HL treatment. Data are normalized to the values obtained with wild-type (WT) plants without any HL exposure. The rates of O2 evolution and consumption of 81 and 180 µmol O2 mg Chl–1 h–1, respectively, in wild-type plants were set at 100%. Data are presented as the means ± SD (n = 3–4). The asterisk marks statistically significant differences (P < 0.05 by Student’s t-test) relative to samples without any HL treatment. To evaluate whether the fbn5 plants lack the capability for protecting the photosynthetic machinery against overexcitation and subsequent damage, we have studied parameters for non-photochemical (NPQ, qE and qI) and photochemical (qP) quenching, and the quantum yield of the Chl photophysical decay pathways (ΦC) in wild-type and fbn5 plants grown under GL conditions (Table 1). NPQ is a parameter for non-photochemical Chl fluorescence quenching, monitoring the apparent rate constant for non-radiative decay (heat loss) from PSII and its antennae. qE and qI, which are major components of NPQ, are related to ΔpH-triggered conformation change in the photosynthetic membranes and photoinhibition/damage to PSII reaction centers, respectively (Ruban 2016). In wild-type plants, at increasing actinic light intensities, NPQ increased drastically, concomitantly with an increase in both qE and qI. In fbn5 plants, NPQ increased from 0.08 to 0.36 with increasing light intensities, to which qI, but not qE, was a major contributor. The fluorescence traces obtained in fbn5 plants, in which the quenching of Chl fluorescence was largely suppressed, were very similar to that in npq4 mutant plants lacking PsbS (Roach and Krieger-Liszkay 2012). These results have suggested that the fbn5 plants lose the capacity for ΔpH-triggered energy dissipation events such as protonation of antenna components, the xanthophyll cycle(s), LHCII rearrangement/aggregation and the formation of the NPQ quencher. Table 1 Various PAM parameters in wild-type and fbn5 plants Light intensity of excitation light (µmol m–2s–1) PAM parameters NPQ qE qI qP ΦC WT 20 0.23 ± 0.07 0.09 ± 0.06 0.14 ± 0.01 0.91 ± 0.04 0.23 ± 0.01 80 0.27 ± 0.02 0.06 ± 0.03 0.20 ± 0.04 0.86 ± 0.01 0.26 ± 0.01 400 1.11 ± 0.14 0.88 ± 0.09 0.24 ± 0.08 0.47 ± 0.01 0.33 ± 0.02 fbn5 20 0.08 ± 0.02 − 0.08 ± 0.03 0.26 ± 0.09 0.78 ± 0.06 80 0.14 ± 0.01 0.015 ± 0.01 0.12 ± 0.02 0.16 ± 0.07 0.79 ± 0.05 400 0.36 ± 0.05 0.032 ± 0.02 0.32 ± 0.06 0.06 ± 0.02 0.71 ± 0.03 Light intensity of excitation light (µmol m–2s–1) PAM parameters NPQ qE qI qP ΦC WT 20 0.23 ± 0.07 0.09 ± 0.06 0.14 ± 0.01 0.91 ± 0.04 0.23 ± 0.01 80 0.27 ± 0.02 0.06 ± 0.03 0.20 ± 0.04 0.86 ± 0.01 0.26 ± 0.01 400 1.11 ± 0.14 0.88 ± 0.09 0.24 ± 0.08 0.47 ± 0.01 0.33 ± 0.02 fbn5 20 0.08 ± 0.02 − 0.08 ± 0.03 0.26 ± 0.09 0.78 ± 0.06 80 0.14 ± 0.01 0.015 ± 0.01 0.12 ± 0.02 0.16 ± 0.07 0.79 ± 0.05 400 0.36 ± 0.05 0.032 ± 0.02 0.32 ± 0.06 0.06 ± 0.02 0.71 ± 0.03 Calculation formulae of PAM parameters are described in the Materials and Methods. Data are means ± SD (n = 3). Table 1 Various PAM parameters in wild-type and fbn5 plants Light intensity of excitation light (µmol m–2s–1) PAM parameters NPQ qE qI qP ΦC WT 20 0.23 ± 0.07 0.09 ± 0.06 0.14 ± 0.01 0.91 ± 0.04 0.23 ± 0.01 80 0.27 ± 0.02 0.06 ± 0.03 0.20 ± 0.04 0.86 ± 0.01 0.26 ± 0.01 400 1.11 ± 0.14 0.88 ± 0.09 0.24 ± 0.08 0.47 ± 0.01 0.33 ± 0.02 fbn5 20 0.08 ± 0.02 − 0.08 ± 0.03 0.26 ± 0.09 0.78 ± 0.06 80 0.14 ± 0.01 0.015 ± 0.01 0.12 ± 0.02 0.16 ± 0.07 0.79 ± 0.05 400 0.36 ± 0.05 0.032 ± 0.02 0.32 ± 0.06 0.06 ± 0.02 0.71 ± 0.03 Light intensity of excitation light (µmol m–2s–1) PAM parameters NPQ qE qI qP ΦC WT 20 0.23 ± 0.07 0.09 ± 0.06 0.14 ± 0.01 0.91 ± 0.04 0.23 ± 0.01 80 0.27 ± 0.02 0.06 ± 0.03 0.20 ± 0.04 0.86 ± 0.01 0.26 ± 0.01 400 1.11 ± 0.14 0.88 ± 0.09 0.24 ± 0.08 0.47 ± 0.01 0.33 ± 0.02 fbn5 20 0.08 ± 0.02 − 0.08 ± 0.03 0.26 ± 0.09 0.78 ± 0.06 80 0.14 ± 0.01 0.015 ± 0.01 0.12 ± 0.02 0.16 ± 0.07 0.79 ± 0.05 400 0.36 ± 0.05 0.032 ± 0.02 0.32 ± 0.06 0.06 ± 0.02 0.71 ± 0.03 Calculation formulae of PAM parameters are described in the Materials and Methods. Data are means ± SD (n = 3). A parameter estimating the fraction of PSII centers in the open state (with QA oxidized representing the redox state of the PQ pool), qP, in wild-type plants was decreased from 0.91 to 0.47 with increasing actinic light intensities. The fbn5 plants were shown to have significantly lower qP; it was 0.26 even at a low actinic light intensity of 20 µmol m–2 s–1 and further decreased to 0.06 at the relatively high actinic light intensity of 400 µmol m–2 s–1. These results indicate that PSII centers in the fbn5 plants are almost closed, or in the reduced state of the PQ pool, even under GL conditions. Furthermore, the values of the parameter ΦC, estimating 1O2 generation by PSII (Ahn et al. 2009; Kasajima et al. 2009), were found to be much greater in fbn5 plants than in wild-type plants at the light intensities examined, suggesting that fbn5 plants generate a considerable amount of 1O2 even under weak light. We next investigated the effects of HL on xanthophylls operating in non-photochemical quenching (Table 2). In HL, violaxanthin (V) is converted into zeaxanthin (Z), via the intermediate antheraxanthin (A), by the enzyme violaxanthin de-epoxidase, resulting in the binding of zeaxanthin to light-harvesting proteins and the subsequent quenching and heat dissipation (Demmig-Adams and Adams 1992, Horton et al. 1996, Jahns and Holzwarth 2012). In wild-type and fbn5/FBN5 plants, the content of zeaxanthin and antheraxanthin was increased by 3.4- and 3.2-fold, respectively, by the HL treatment, and the de-epoxidation status of the xanthophyll cycle pigments [i.e. (A + Z)/(V + A + Z)] was increased. This indicates that a xanthophyll cycle functions normally under HL conditions in wild-type and fbn5/FBN5 plants. In the fbn5 plants, the contents of zeaxanthin and antheraxanthin were slightly greater compared with those in the wild-type plants under GL conditions, while they were increased by 1.4-fold by the HL treatment, but the de-epoxidation status was not increased substantially. The fbn5 plants showed a lower Chl content per unit of leaf area (Supplementary Fig. S4). These results suggest that in the fbn5 plants photoinhibition occurs even under GL conditions and that a xanthophyll cycle inefficiently functions in non-photochemical quenching processes. Table 2 Pigment contents in leaves of wild-type, fbn5 and fbn5/FBN5 plants Pigment content (nmol g–1 FW) Neo Vio Ant Zea Lut β-car Chl a + b Chl a/b (A + Z)/ (V + A + Z) GL WT 47.8 ± 5.80 62.6 ± 9.26 2.79 ± 0.45 3.98 ± 1.56 231 ± 26.1 96.4 ± 6.72 1,742 ± 156 3.31 ± 0.03 0.10 ± 0.03 fbn5 75.1 ± 3.55 124 ± 7.04 8.88 ± 4.10 13.3 ± 5.80 329 ± 45.3 129 ± 10.2 2,690 ± 454 2.64 ± 0.16 0.15 ± 0.03 fbn5/FBN5 65.0 ± 5.2 72.5 ± 3.32 3.35 ± 0.41 7.4 ± 2.20 305 ± 16.2 113 ± 4.9 2,069 ± 166 3.16 ± 0.04 0.13 ± 0.03 HL WT 64.1 ± 12.9 48.2 ± 10.3 13.2 ± 5.16 9.84 ± 7.20 328 ± 63.8 112 ± 7.66 1,909 ± 181 3.10 ± 0.05 0.33 ± 0.07 fbn5 72.2 ± 1.22 74.4 ± 0.82 22.3 ± 3.02 7.67 ± 3.80 356 ± 14.9 97.5 ± 12.4 2,402 ± 336 2.67 ± 0.15 0.28 ± 0.04 fbn5/FBN5 75.8 ± 1.8 46.8 ± 3.71 13.35 ± 2.87 21.5 ± 13.6 389 ± 67.0 152 ± 2.8 2,492 ± 235 3.13 ± 0.04 0.42 ± 0.07 Pigment content (nmol g–1 FW) Neo Vio Ant Zea Lut β-car Chl a + b Chl a/b (A + Z)/ (V + A + Z) GL WT 47.8 ± 5.80 62.6 ± 9.26 2.79 ± 0.45 3.98 ± 1.56 231 ± 26.1 96.4 ± 6.72 1,742 ± 156 3.31 ± 0.03 0.10 ± 0.03 fbn5 75.1 ± 3.55 124 ± 7.04 8.88 ± 4.10 13.3 ± 5.80 329 ± 45.3 129 ± 10.2 2,690 ± 454 2.64 ± 0.16 0.15 ± 0.03 fbn5/FBN5 65.0 ± 5.2 72.5 ± 3.32 3.35 ± 0.41 7.4 ± 2.20 305 ± 16.2 113 ± 4.9 2,069 ± 166 3.16 ± 0.04 0.13 ± 0.03 HL WT 64.1 ± 12.9 48.2 ± 10.3 13.2 ± 5.16 9.84 ± 7.20 328 ± 63.8 112 ± 7.66 1,909 ± 181 3.10 ± 0.05 0.33 ± 0.07 fbn5 72.2 ± 1.22 74.4 ± 0.82 22.3 ± 3.02 7.67 ± 3.80 356 ± 14.9 97.5 ± 12.4 2,402 ± 336 2.67 ± 0.15 0.28 ± 0.04 fbn5/FBN5 75.8 ± 1.8 46.8 ± 3.71 13.35 ± 2.87 21.5 ± 13.6 389 ± 67.0 152 ± 2.8 2,492 ± 235 3.13 ± 0.04 0.42 ± 0.07 Pigment measurements were performed before and after exposure of GL (80 µmol m–2 s-1)-grown plants to HL (800 µmol m–2 s–1) for 4 h. Data are presented as the means ± SD (n = 4–5). Neo, neoxanthin; Vio, violaxanthin (V); Ant, antheraxanthin (A); Zea, zeaxanthin (Z); Lut, lutein; β-car, β-carotene. Table 2 Pigment contents in leaves of wild-type, fbn5 and fbn5/FBN5 plants Pigment content (nmol g–1 FW) Neo Vio Ant Zea Lut β-car Chl a + b Chl a/b (A + Z)/ (V + A + Z) GL WT 47.8 ± 5.80 62.6 ± 9.26 2.79 ± 0.45 3.98 ± 1.56 231 ± 26.1 96.4 ± 6.72 1,742 ± 156 3.31 ± 0.03 0.10 ± 0.03 fbn5 75.1 ± 3.55 124 ± 7.04 8.88 ± 4.10 13.3 ± 5.80 329 ± 45.3 129 ± 10.2 2,690 ± 454 2.64 ± 0.16 0.15 ± 0.03 fbn5/FBN5 65.0 ± 5.2 72.5 ± 3.32 3.35 ± 0.41 7.4 ± 2.20 305 ± 16.2 113 ± 4.9 2,069 ± 166 3.16 ± 0.04 0.13 ± 0.03 HL WT 64.1 ± 12.9 48.2 ± 10.3 13.2 ± 5.16 9.84 ± 7.20 328 ± 63.8 112 ± 7.66 1,909 ± 181 3.10 ± 0.05 0.33 ± 0.07 fbn5 72.2 ± 1.22 74.4 ± 0.82 22.3 ± 3.02 7.67 ± 3.80 356 ± 14.9 97.5 ± 12.4 2,402 ± 336 2.67 ± 0.15 0.28 ± 0.04 fbn5/FBN5 75.8 ± 1.8 46.8 ± 3.71 13.35 ± 2.87 21.5 ± 13.6 389 ± 67.0 152 ± 2.8 2,492 ± 235 3.13 ± 0.04 0.42 ± 0.07 Pigment content (nmol g–1 FW) Neo Vio Ant Zea Lut β-car Chl a + b Chl a/b (A + Z)/ (V + A + Z) GL WT 47.8 ± 5.80 62.6 ± 9.26 2.79 ± 0.45 3.98 ± 1.56 231 ± 26.1 96.4 ± 6.72 1,742 ± 156 3.31 ± 0.03 0.10 ± 0.03 fbn5 75.1 ± 3.55 124 ± 7.04 8.88 ± 4.10 13.3 ± 5.80 329 ± 45.3 129 ± 10.2 2,690 ± 454 2.64 ± 0.16 0.15 ± 0.03 fbn5/FBN5 65.0 ± 5.2 72.5 ± 3.32 3.35 ± 0.41 7.4 ± 2.20 305 ± 16.2 113 ± 4.9 2,069 ± 166 3.16 ± 0.04 0.13 ± 0.03 HL WT 64.1 ± 12.9 48.2 ± 10.3 13.2 ± 5.16 9.84 ± 7.20 328 ± 63.8 112 ± 7.66 1,909 ± 181 3.10 ± 0.05 0.33 ± 0.07 fbn5 72.2 ± 1.22 74.4 ± 0.82 22.3 ± 3.02 7.67 ± 3.80 356 ± 14.9 97.5 ± 12.4 2,402 ± 336 2.67 ± 0.15 0.28 ± 0.04 fbn5/FBN5 75.8 ± 1.8 46.8 ± 3.71 13.35 ± 2.87 21.5 ± 13.6 389 ± 67.0 152 ± 2.8 2,492 ± 235 3.13 ± 0.04 0.42 ± 0.07 Pigment measurements were performed before and after exposure of GL (80 µmol m–2 s-1)-grown plants to HL (800 µmol m–2 s–1) for 4 h. Data are presented as the means ± SD (n = 4–5). Neo, neoxanthin; Vio, violaxanthin (V); Ant, antheraxanthin (A); Zea, zeaxanthin (Z); Lut, lutein; β-car, β-carotene. Based on a quantitative analysis of α-tocopherol and PQ-9, the amount of α-tocopherol in chloroplasts isolated from fbn5 plants was twice as high as in wild-type plants, while the amount of PQ-9 in chloroplasts isolated from fbn5 plants was 5-fold lower in wild-type plants (Fig. 6). The amounts of these lipids were gradually decreased during HL treatment in the wild-type and fbn5 plants. The ratio of reduced PQ-9 to total PQ-9 was increased from 50% to 60% by the 4 h HL treatment in wild-type plants, whereas it remained significantly high ( >90%) in fbn5 plants. Fig. 6 View largeDownload slide Quantitative determination of α-tocopherol, PQ-9 and plastoquinol-9. Lipids were extracted from leaves of 5- to 6-week-old plants during 4 h HL treatment, and analyzed with HPLC as described in the Materials and Methods. Data are presented as the means ± SD (n =3). Fig. 6 View largeDownload slide Quantitative determination of α-tocopherol, PQ-9 and plastoquinol-9. Lipids were extracted from leaves of 5- to 6-week-old plants during 4 h HL treatment, and analyzed with HPLC as described in the Materials and Methods. Data are presented as the means ± SD (n =3). FBN5 possessing a chloroplast transit signal at the N-terminus was previously reported to be present in the stroma, by mass spectroscopy analyses of the total leaf, thylakoid and stroma (Lundquist et al. 2012), and immunoblots with an anti-green fluorescent protein (GFP) antibody in transformed leaf protoplasts expressing the FBN5–GFP fusion proteins (Kim et al. 2015). We pursued the clarification of the subcellular localization and accumulation of FBN5 by quantitative immunoblot analysis of simply fractionated chloroplasts isolated from 6-week-old leaves with polyclonal antibodies to epitopes in the C-terminus of FBN5. Fig. 7 clearly shows the band with the predicted molecular weight (23.9 kDa) of FBN5 in only the stromal fraction; in the thylakoid fraction, no band with a molecular weight similar to FBN5 was detected. This result indicates that a FBN5 mature protein is localized in the stroma. When the plants were subjected to HL or low temperature, significant accumulation of FBN5 in wild-type plants was observed (Fig. 7B, C). The exposure of the plants to HL for 4 h did not induce distinct accumulation of FBN5, but 24 h HL exposure stimulated the accumulation of FBN5 by about 50% as compared with GL exposure. The exposure of the plants to low temperature (8°C) also enhanced the accumulation of FBN5, which was diminished by the combination of HL and low temperature (Fig. 7C). The accumulation of FBN-1a/b, -7b and -8 in PGs was reported to be increased by the HL treatment (Ytterberg et al. 2006). These results indicate that the HL or cold treatment modulates the rates of the synthesis and degradation of FBN5 in a different manner. Fig. 7 View largeDownload slide Immunodetection of FBN5 in stroma and thylakoid fractions. (A) Intact chloroplasts from wild-type, fbn5 and fbn5/FBN5 plants were separated into stromal and thylakoid fractions; equivalent amounts (10 µg of protein) were subject to SDS–PAGE, and then Western blotting using antibodies against FBN5, D1 (as a marker for the thylakoid membrane) and RbcL (as a stromal marker). C, intact chloroplasts; T, thylakoids; S, stroma. (B, C) Immunoblot analysis and ImageJ densitometry analysis of FBN5 in the crude stroma. Chloroplasts were prepared from rosette leaves from wild-type plants exposed to HL for 4 or 24 h at 4 or 23°C. Data are normalized to the values obtained under GL conditions at 23°C. Data are presented as the means ± SD (n = 3). Asterisks indicate a statistically significant difference relative to the values obtained under GL conditions at 23°C (P < 0.05 by Student’s t-test). Experiments in (B) and (C) were done separately with different preparations. Fig. 7 View largeDownload slide Immunodetection of FBN5 in stroma and thylakoid fractions. (A) Intact chloroplasts from wild-type, fbn5 and fbn5/FBN5 plants were separated into stromal and thylakoid fractions; equivalent amounts (10 µg of protein) were subject to SDS–PAGE, and then Western blotting using antibodies against FBN5, D1 (as a marker for the thylakoid membrane) and RbcL (as a stromal marker). C, intact chloroplasts; T, thylakoids; S, stroma. (B, C) Immunoblot analysis and ImageJ densitometry analysis of FBN5 in the crude stroma. Chloroplasts were prepared from rosette leaves from wild-type plants exposed to HL for 4 or 24 h at 4 or 23°C. Data are normalized to the values obtained under GL conditions at 23°C. Data are presented as the means ± SD (n = 3). Asterisks indicate a statistically significant difference relative to the values obtained under GL conditions at 23°C (P < 0.05 by Student’s t-test). Experiments in (B) and (C) were done separately with different preparations. To evaluate how FBN5 functions in the processes of acclimation, we have studied the effects of FBN5 on the expression of FBN family genes and stress-related genes (Fig. 8). These genes were selected from the FBN family and major genes of the following pathways which were previously reported to have more involvement with the FBN family; photosynthesis, PQ-9 and α-tocopherol biosynthesis, the xanthophyll cycle and carotenoid biosynthesis, ABA biosynthesis and jasmonate biosynthesis (Fig. 8A–F). In wild-type plants, the expression of FBN5 was increased by 4-fold in the 1 h HL treatment, but was subsequently decreased gradually by further HL treatment. FBN-1a, -1b, -2 and -4 are reported probably to be required for resistance to light/cold stress. Expression of FBN1a and FBN1b was significantly increased under HL in wild-type plants, while the expression of FBN2 and FBN4 was transiently increased at the beginning of HL treatment. The fbn5 plants showed the suppression of gene expression of FBN-1a, -1b and -2 during HL treatment, with some differences in the extent of their expression (Fig. 8A). These results suggest that the functional link between FBN5 and major members of the FBN family exists. Fig. 8 View largeDownload slide Effects of HL on gene expression of FBN family, photosynthesis- and stress-related genes. Transcript levels of the following genes in mature leaves of 6-week-old wild-type (circle) and fbn5 (triangle) plants were measured during 4 h HL treatment: (A) FBN5, FBN1a, FBN1b, FBN2 and FBN4. (B) PsbA, PSII subunit A (D1); PsbB, PSII subunit B (CP47); LHCB4.2, PSII light-harvesting complex gene 4.2; STN7/8, state transition 7/8. (C) SPS1/2, solanesyl diphosphate synthase 1/2; HST, homogentisate solanesyltransferase; HPT, homogentisate phytyltransferase. (D) PsbS, PSII subunit S; ZDS, ζ-carotene desaturase; VDE, violaxanthin de-epoxidase; ZEP, zeaxanthin epoxidase. (E) NCED3, 9-cis-epoxycarotenoid dioxygenase 3. (F) LOX2, lipoxygenase 2; AOS, allene oxide synthase; AOC2, allene oxide cyclase 2. mRNA levels were determined by quantitative real-time PCR using gene-specific primers (Supplementary Table S1), and were shown relative to the expression of ACT2 in each sample. Data are presented as the means ± SE (n =3–4). The genes up- and down-regulated by the HL treatment in fbn5 plants compared with the wild type are highlighted in yellow and blue, respectively. Fig. 8 View largeDownload slide Effects of HL on gene expression of FBN family, photosynthesis- and stress-related genes. Transcript levels of the following genes in mature leaves of 6-week-old wild-type (circle) and fbn5 (triangle) plants were measured during 4 h HL treatment: (A) FBN5, FBN1a, FBN1b, FBN2 and FBN4. (B) PsbA, PSII subunit A (D1); PsbB, PSII subunit B (CP47); LHCB4.2, PSII light-harvesting complex gene 4.2; STN7/8, state transition 7/8. (C) SPS1/2, solanesyl diphosphate synthase 1/2; HST, homogentisate solanesyltransferase; HPT, homogentisate phytyltransferase. (D) PsbS, PSII subunit S; ZDS, ζ-carotene desaturase; VDE, violaxanthin de-epoxidase; ZEP, zeaxanthin epoxidase. (E) NCED3, 9-cis-epoxycarotenoid dioxygenase 3. (F) LOX2, lipoxygenase 2; AOS, allene oxide synthase; AOC2, allene oxide cyclase 2. mRNA levels were determined by quantitative real-time PCR using gene-specific primers (Supplementary Table S1), and were shown relative to the expression of ACT2 in each sample. Data are presented as the means ± SE (n =3–4). The genes up- and down-regulated by the HL treatment in fbn5 plants compared with the wild type are highlighted in yellow and blue, respectively. The expression patterns of photosynthesis-related genes under HL stress were similar in wild-type and fbn5 plants: the peaks of expression of PsbA, PsbB and LHCB4.2 genes appeared at early stages of HL treatment, whereas STN7 and STN8 whose products phosphorylate LHCII and PSII core proteins, respectively, were almost constantly expressed (Fig. 8B). In particular, the lack of FBN5 suppressed the expression of PsbA and PsbB during HL treatment. The fbn5 plants under GL conditions showed almost identical levels of expression of SPS1/2 and HST genes that are involved in PQ-9 biosynthesis (Fig. 8C) (Kim et al. 2015). Under HL stress conditions, the expression of SPS1/2 genes was maximized at the early stages of the stress, a phenomenon which became more remarkable in the fbn5 plants. In contrast, the expression of HST remained unchanged during the HL treatment in both wild-type and fbn5 plants. The expression of HPT, encoding a key enzyme of α-tocopherol biosynthesis, was gradually increased during the HL treatment, but it remained almost constant at low levels in fbn5 plants. These results suggest that FBN5 has implications for α-tocopherol biosynthesis as well as PQ-9 biosynthesis. As shown in Fig. 5A and Table 1, fbn5 plants were hypersensitive to light, resulting in strengthening of photoprotection by dissipation of excess light energy such as via the xanthophyll cycle. The expression of PsbS and the genes involved in the xanthophyll cycle was studied under HL treatment (Fig. 8D). The expression levels of these genes in fbn5 plants were significantly enhanced at all or some of the treatment times examined. This was coincident with the results in Table 2 showing that the amounts of xanthophylls and carotenoids were greater in fbn5 plants than in the wild type under both GL and HL conditions. These results strongly support that the dissipation of excess light energy is accelerated in a proactive manner in fbn5 plants. We further examined the expression of genes involved in ABA signal transduction and jasomonate biosynthesis, which were reported to be functionally related to FBN1a, FBN1b or FBN2 (Fig. 8E, F) (Yang et al. 2006, Youssef et al. 2010). In fbn5 plants, all three genes of jasmonate biosynthesis and NCED of ABA biosynthesis were expressed repressively under both GL and HL conditions. From these results, FBN5 as well as FBN1a/1b/2 is suggested to function in tolerance against environmental stresses directly or indirectly. Discussion The FBN family with a predicted lipocalin motif is a small hydrophobic ligand-binding protein family. Recently, Kim et al. (2015) have reported that FBN5 is required for PQ-9 biosynthesis through its interaction with SPS1/2 and that FBN5-deficient plants were sensitive to cold stress. Some of the FBN family proteins have also been reported to be accumulated under abiotic/biotic stress conditions, and are involved in the response to these stresses as follows: (i) FBN levels (most probably FBN1a and FBN1b) were correlated with protection against photoinhibition under HL, and the ABA response regulators ABI1 and ABI2 associating with FBN1a seemed to control fibrillin expression that was involved in mediating ABA-induced photoprotection (Yang et al. 2006); (ii) FBN1-2 knockdown plants contained less triacylglycerol under high light/cold stress, resulting in a decrease in expression of several jasmonate-induced genes (Youssef et al. 2010); (iii) in the FBN4-knockdown and -knockout plants, the amount of PQ-9 was reported to be drastically decreased, and was susceptible to HL (Singh et al. 2010, Singh et al. 2012). According to the relative protein abundances acquired from the Arabidopsis abundance data sets aggregated in the PaxDb database (Baerenfaller et al. 2012, Wang et al. 2012), FBN5 is one of the minor proteins in the FBN family, as already reported (Lundquist et al. 2012); the protein abundance in leaf of FBN5 was much lower than those of FBN-1a, -1b, -4, -2, -7b, -6 and -3a, and was comparable with those of FBN-10, -8, -7a and -3b. The FBN5 protein abundance in leaf was also comparable with that of SPS1/2 that have been reported to interact physically with FBN5. FBN5 is expressed organ specifically; the protein abundance of FBN5 in individual organs was in the following order of highest to lowest: juvenile leaf > cotyledon > leaf > root > carpel, flower, flower bud, pollen, seed, shoot and silique. Such organ specificity in protein abundance was similar to that in mRNA abundance in this study (Fig. 2B), although the mRNA abundance in flower was considerably higher. The FBN5 protein was located in the stroma, as already reported (Lundquist et al. 2012), which was not affected by HL treatment (Supplementary Fig. S5). While FBN5 is expressed at a low level with respect to protein abundance, the dysfunction of this gene led to critical changes in structural and functional properties of plants as shown by the following evidence revealed by this study. (i) The fbn5 plants grown even under GL conditions at 80 µmol m–2 s–1 showed a drastic decrease in electron transfer activities around PSII, with little change in electron transfer activities around PSI, a phenomenon which was exaggerated under HL stress (Fig. 5; Table 1). (ii) The accumulation of PQ-9 was severely suppressed in fbn5 plants, and both photoactive and non-photoactive PQ pools were almost reduced based on the HPLC analyses and the qP component of NPQ (Fig. 6; Table 1). The content of carotenoids such as xanthophylls, β-carotene and lutein acting as antioxidants, as well as PQ-9, was increased rather than unchanged or decreased (Table 2). These findings were supported by the evidence that there was a remarkable difference in PG osmiophilicity between wild-type and fbn5 plants (Fig. 3G, H, J, K;Supplementary Fig. S6); chloroplasts in fbn5 plants mainly contained electron-transparent (non-osmiophilic, or white) PGs, while wild-type chloroplasts contained many electron-opaque (osmiophilic, or black) PGs. Two types of PGs differing in their osmiophilicity were found by Singh et al. (2010), which is thought to result from the reactivity of osmium oxides with certain biological materials such as prenyl quinones, carotenoids and triacylglycerides. Our findings suggest that PG constituents of antioxidants such as PQ-9, carotenoids and triacylglycerides are drastically reduced, probably reflecting the increased stress sensitivity of the fbn5 plants. Similar results were reported in fbn4-knockdown mutants (Singh et al. 2010). (iii) PAM analyses with fbn5 plants showed that NPQ was much lower than that in wild-type plants although NPQ was increased by the HL treatment; qE, a parameter for short-term control, hardly contributed to the total NPQ; and qI, a parameter for long-term control, was a major contributor of NPQ (Table 1). (iv) Deletion of FBN5 had an opposite influence on the expression of FBN family genes and stress-related genes during the HL treatment (Fig. 8). The fbn5 plants showed the suppression of expression of FBN-1a, -1b and -2 during the HL treatment, as well as the suppression of genes of the PSII reaction center (PsbA and PsbB) and major genes for jasmonate and ABA biosynthesis (LOX2, AOS, AOC2 and NCED3). On the other hand, in the fbn5 plants, the expression of genes involved in PQ-9 biosynthesis (SPS1 and SPS2) and in xanthophyll cycle/biosynthesis (PsbS, ZDS, VDE and ZEP) were considerably enhanced, although their expression patterns were different during HL treatment. (v) The ultrastructure of thylakoids in fbn5 plants was drastically changed: thylakoid membranes were swollen and fragmented, and their grana were unstacked and transformed into loose and disordered structures (Fig. 3H), a phenomenon which was reported with the thylakoid membranes induced by light stress (Chuartzman et al. 2008, Herbstová et al. 2012) and the chloroplasts which were impaired in the reducing sides of PSII (Hagio et al. 2002). Furthermore, in the fbn5 plants, HL treatment caused a marked change in the ultrastructure of chloroplasts in that the grana became more fragmentated (Fig. 3K), although hardly any change in the ultrastructure was observed in the wild-type and fbn5/FBN5 plants (Fig. 3J, L). These structural changes have not been reported with the thylakoid membranes in other fbn mutants such as the fbn1a/fbn1b-knockout mutants of Arabidopsis (Gamez-Arjona et al. 2014), FBN1-2 RNAi (RNA interference) lines of Arabidopsis (Youssef et al. 2010) and FBN4 RNAi lines of apple trees (Singh et al. 2010). The characteristics of fbn5 mutant plants described above can be explained mostly by a decline in the ability to accumulate PQ-9, which causes the limitation of electron transfer from PSII to the Cyt b6f complex to produce ROS (1O2, O2·– and H2O2) by giving excess electrons on the reducing side of PSII to oxygen molecules (Fischer et al. 2013). The redox state of quinone acceptors of PSII are shifted to a reduced state by such excess supply of electrons, which increases the probability of 1O2 and O2·– generation by the charge recombination of P680+ pheophytin– to 3P680 (Rutherford and Krieger-Liszkay 2001) and the reduction of molecular O2 via plastid terminal oxidase (PTOX) (Yu et al. 2014), respectively. Indeed, as shown in Table 1, fbn5 plants have shown greater values of a parameter ΦC which represents the possibility of 1O2 generation by PSII. To protect plants from such photodamage by ROS, plants evolutionarily obtain the mechanisms for both short- and long-term control of the input of light energy to photosynthetic reaction centers (Foyer et al. 2012, Derks et al. 2015, Ruban 2016). In this study, the deletion of FBN5, which represented misconducted the short-term control such as state transitions and xanthophyll cycle which should occur within minutes. Furthermore, FBN5 also did the long-term control such as PSII repair and plant development which should occur on the order of hours through the processes of light-dependent regulation of gene expression. With regard to the relationship between the PQ-9 abundance and phototolerance, Ksas et al. (2015) have clarified that the SPS1-overexpressing lines, which specifically accumulate PQ-9 and its derivative plastochromanol-8, were much more resistant to HL stress than the wild type, showing marked decreases in leaf bleaching, lipid peroxidation and PSII photoinhibition under excess light. Consequently, the fbn5 plants elicited a hypersensitive response in light, which may result from the low yield of PQ-9 and the shift of the reduced form of PQ-9. Our results strongly support the conclusion by Kim et al. (2015) that FBN5 functions as a structural component involved in the biosynthesis of PQ-9 by forming FBN5-B/SPS homodimeric complexes. Besides this function of FBN5, we propose another function in the light stress response. The transcriptional response to increased 1O2 production is clearly different from the response to other ROS such as O2·– and H2O2 (Op den Camp et al. 2003, Dietz et al. 2016). The signal transduction of 1O2 from thylakoids to the cytoplasm/nucleus via the stroma, one type of chloroplast retrograde signaling, forms an integral part of a complex signaling network that is connected to other signaling routes involving phytohormones such as jasmonate (Tikkanen et al. 2014, Dietz et al. 2016, Shumbe et al. 2016). The 1O2 generated mainly by PSII is well known to up-regulate the transcription of jasmonate-responsive genes and genes involved in the biosynthesis of jasmonate. Indeed, in the wild-type plants in this study, the HL treatment markedly enhanced the expression of genes involved in the biosynthesis of jasmonate (LOX2, AOS and AOC2) (Fig. 8F). However, in fbn5 plants, although the production of 1O2 is considered to be elevated under HL and even GL conditions (Table 1), the expression of these genes was down-regulated. A similar phenomenon was reported with Arabidopsis mutants in which FBN-1a, -1b and -2 are all down-regulated (Youssef et al. 2010); FBNs 1a/1b/2 are located in PGs, while FBN5 is found in the stroma. From these results, we speculate that FBN5, as well as FBNs 1a/1b/2, functions as a transmitter of the 1O2 signal pathway in a stromal region. Recently, specific sensors for increased 1O2 have been detected in the thylakoids or via 1O2 reaction products in the stroma; they are oxidation products of carotenoids (such as β-cyclocitral) and lipids (such as the oxylipins 13-hydroxy octadecadienoic acid and/or 13-keto-octadecatrienoic acid, linolenic acid derivatives and precursors of jasmonate) and two chloroplastic proteins, EXECUTER1 (EX1) and EX2 (Wagner et al. 2004, Dietz et al. 2016). FBN5 possesses a lipocalin motif that can bind a small hydrophobic molecule. Furthermore, Malnoë et al. (2018) have reported that the plastid lipocalin, LCNP, is required for sustained photoprotective energy dissipation. Although we were unable to observe a partial translocation of the FBN5 protein from the stroma to thylakoid membranes under the light stress conditions (data not shown), FBN5 is suggested to carry such signaling compounds in the stroma. Further studies will be needed to unveil a functional role for FBN5 in the 1O2 signaling pathway in the chloroplast. Materials and Methods Plant materials and growth conditions All Arabidopsis thaliana (L) Heynh. mutants used in this work were of the Col-0 background. The knockout mutant fbn5 (SALK_064597) was acquired from T-DNA insertion lines of the ABRC. Surface-sterilized wild-type, mutant and transgenic seeds were plated onto Murashige and Skoog (MS) medium with 1% sucrose. The plated seeds were vernalized at 4°C for 3 d in the dark to synchronize germination and then transferred to a growth chamber at 23°C under a 12 h light:12 h dark cycle using GL (photon flux density of 80 µmol m–2 s–1). Two-week-old seedlings were then grown in Jiffy-7 33 mm soil (Jiffy International AS) for 4 weeks under the same conditions as described above. Activation-tagging and freezing-tolerant mutant selection Transformation of calli was performed as described by Otsubo et al. (2007), which was based on the method by Akama et al. (1992). Transformed calli were cooled to −14°C in a freezing bath (NCB-3200, EYELA). The calli were thawed and transferred to shoot-inducing medium (SIM; MS medium supplemented with IAA and trans-zeatin). Surviving transformants were transferred to new SIM plates and ascribed as candidates for freezing-tolerant mutants. RNA extraction, DNA extraction and PCR Total RNA from Arabidopsis leaves was isolated using TRIzol (Invitrogen) according to the manufacturer’s protocol. A 1 μg aliquot of RNA was used as a template for first-strand cDNA synthesis using ReverTra Ace qPCR RT Master Mix with gDNA Remover (Toyobo). Genomic DNA was isolated using the DNeasy Plant Miniprep kit (Qiagen). PCR was performed using EX Taq (TAKARA) or KOD FX (Toyobo). Primer sequences and descriptions are provided in Supplementary Table S1. Complementation of the Arabidopsis fbn5 mutation The full-length FBN5 coding sequences (FBN5-259 and FBN5-273) were amplified from cDNA obtained from Arabidopsis leaves. The amplified products were XbaI/BamHI digested and cloned into the binary vector pCAM35S [the CaMV 35S promoter and the NOS terminator from pBI121 were inserted into the multiple cloning site (MCS) of pCAMBIA1300]. The resulting vectors were then introduced into competent Agrobacterium tumefaciens cells (GV3101) by the freeze–thaw method (Chen et al. 1994). The fbn5 plants were transformed with the Agrobacterium described above, using the floral dip transformation method (Clough and Bent 1998), and selected by their resistance to hygromycin B. Real-time RT–PCR Quantitative RT–PCR (qRT-PCR) was performed using gene-specific primers (Supplementary Table S1) on a LightCycler 480 system using LightCycler 480 SYBR Green I master mix (Roche Diagnostics). The amplification efficiencies of all primers were between 1.8 and 2.2 for all samples tested. We determined the crossing point of each sample using the Second Derivative Maximum method of the software, and calculated relative mRNA levels using the relative standard curve method. The expression of each gene was normalized to the expression of Actin2. Electron microscopy analysis Fully expanded leaves from plants cultured under a 12 h light/12 h dark photoperiod were collected at the indicated times. Small isosceles triangle pieces (base 2 mm and height 5 mm) of leaves were cut with a razor blade and immediately fixed in 1% glutaraldehyde in 20 mM Na-cacodylate buffer, pH 7.4, at 4°C overnight. After washing with Na-cacodylate buffer, the samples were fixed with 1% OsO4 at 4°C for 1.5 h, dehydrated in a graded series of ethanol and then embedded in Epon 812. Ultrathin (70 nm) sections were cut with an Ultracut EM UC7 microtome (Leica) fitted with a diamond knife. Sections were contrasted with 2% aqueous uranyl acetate and lead citrate (Reynolds 1963). Observations were performed using a JEM 1400 Plus transmission electron microscope (Jeol) at 120 kV, equipped with a fully integrated high-resolution camera system (8 million pixel CCD camera). Different sections from at least three different leaf samples were analyzed. Isolation and subfractionation of intact chloroplasts Intact chloroplasts were isolated from 1–2 g of Arabidopsis rosette leaves by the method of Aronsson and Jarvis (2002). To isolate thylakoids and crude stroma from the intact chloroplasts, intact chloroplasts were lysed in a hypertonic medium (50 mM HEPES, pH 8.0, 5 mM MgCl2) for 30 min on ice, and thylakoid membranes were collected by centrifugation at 10,000×g for 10 min. The resultant supernatant, the crude stromal fraction, was further concentrated with an Amicon Ultra-4 10 K centrifugal filter device (Merck). Protein amounts were determined using the Micro BCA protein assay kit (Thermo Fisher Scientific). Immunoblot analysis Proteins were transferred from an SDS–polyacrylamide gel onto a PVDF membrane by electroblotting using a Trans-Blot SD transfer cell (Bio-Rad). Blots were probed with rabbit anti-RbcL (Agrisera) (1:20,000 dilution), anti-PsbA (kindly gifted by Professor Y.Yamamoto, Okayama University) (1:20,000 dilution) or anti-FBN5 (Sigma-Aldrich) (1 µg ml–1) followed by a horseradish peroxidase-conjugated goat anti-rabbit IgG serum (Santa Cruz Biotechnology), and detected using ECL Advance Western Blotting Reagent (GE Healthcare). Chemiluminescence was visualized using a LuminoGraph II (Atto) apparatus running ImageSaver6 software (Atto). Native green gel electrophoresis Separation of different Chl–protein complexes of isolated thylakoid membranes was performed following the method described previously (Allen and Staehelin 1991). The samples of isolated thylakoid membranes were solubilized for 30 min on ice in a buffer solution containing 0.45% (w/v) n-octyl-β-d-glucoside, 0.45% (w/v) n-dodecyl-β-d-maltoside, 0.1% (w/v) lithium dodecyl sulfate and 10% (w/v) sucrose to adjust the ratio of total non-ionic detergents to Chl at 20:1 (w/w). The unsolubilized fragments were removed by centrifugation and the supernatant obtained was resolved on a 7% separating polyacrylamide gel. Measurement of photosynthetic parameters Electron transport activities of PSI and PSII were measured by evolution and consumption, respectively, of oxygen with a Clark-type oxygen electrode at 23°C unless otherwise indicated (Izawa 1980). PSII maximum efficiency and parameters for photochemical and non-photochemical quenching were obtained by use of a Chl fluorometer Mini-PAM (Walz) at 23°C. Saturation pulse Chl fluorescence yield parameters (Fo, Fm, Fs, Fm', Fm'' and Fo'') were recorded as described in the literature (Avenson et al. 2004, Baker et al. 2007, Baker 2008). Before measuring fluorescence emission, the leaves were dark adapted for 10 min. The PSII maximum quantum yield (Fv/Fm) was obtained from Chl a fluorescence as (Fm – Fo)/Fm, where Fo is the initial Chl fluorescence level and Fm is the maximal fluorescence level, determined with a 0.8 s saturation pulse of approximately 5,000 μmol m–2 s–1. Fs is the steady-state fluorescence level under actinic illumination at different photon flux densities (20, 80 and 400 µmol m–2 s–1), while Fm' and Fm'' are the maximum fluorescence in 5 min actinic illumination and in the subsequent 5 min dark recovery, respectively. Fluorescence parameters employed in this study were calculated as follows: qP = (Fm' – Fs)/(Fm' – Fo'), NPQ = (Fm – Fm')/Fm'), qE = Fm/Fm' − Fm/Fm'', qI = Fm/Fm'' – 1 and ΦC = Fs/Fm (Krause and Jahns 2003, Baker et al. 2007, Ahn et al. 2009, Ruban 2016). More than three measurements were made from three different plants for each plant type. Values are represented as means ± SD (n = 3). Lincomycin treatment Detached leaves were harvested from approximately 5-week-old plants, and were put into 20 ml of Tris-buffered saline (TBS) containing 5 mM lincomycin and 0.2% (v/v) Tween-20. Lincomycin was infiltrated into the leaves by a water aspirator for 90 s. Leaf discs treated with lincomycin were immediately placed on wet filter papers and irradiated with two different light intensities (80 and 400 μmol m–2 s–1), prior to PAM measurements. Analysis of pigments, PQ-9 and α-tocopherol For pigment analysis, frozen tissues were ground in liquid nitrogen, and the pigments were extracted in 85% acetone and analyzed with HPLC using an Agilent 1100 HPLC system, basically according to the method of García-Plazaola and Becerril (1999). To analyze PQ-9 and α-tocopherol, the lipids were extracted from frozen leaves by the method of Kruk and Trebst (2008). Supplementary Data Supplementary data are available at PCP online. Funding This work was supported by the Fukuoka Industry, Science & Technology Foundation. Acknowledgments We would like to acknowledge Drs. Yasusi Yamamoto (Okayama University) and Kensuke Kusumi (Kyushu University) for critical reading of the manuscript. We also acknowledge Dr. Shun Hamada (Fukuoka Women’s University) for technical advice on electron microscopy. Disclosures The authors have no conflicts of interest to declare. References Ahn T.K. , Avenson T.J. , Peers G. , Li Z. , Dall’osto L. , Bassi R. ( 2009 ) Investigating energy partitioning during photosynthesis using an expanded quantum yield convention . Chem. Phys . 357 : 151 – 158 . Google Scholar CrossRef Search ADS Akama K. , Shiraishi H. , Ohta S. , Nakamura K. , Okada K. , Shimura Y. ( 1992 ) Efficient transformation of Arabidopsis thaliana: comparison of the efficiencies with various organs, plant ecotypes and Agrobacterium strains . Plant Cell Rep . 12 : 7 – 11 . 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Functional Role of Fibrillin5 in Acclimation to Photooxidative Stress

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© The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com
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Abstract

Abstract The functional role of a lipid-associated soluble protein, fibrillin5 (FBN5), was determined with the Arabidopsis thaliana homozygous fbn5-knockout mutant line (SALK_064597) that carries a T-DNA insertion within the FBN5 gene. The fbn5 mutant remained alive, displaying a slow growth and a severe dwarf phenotype. The mutant grown even under growth light conditions at 80 µmol m–2 s–1 showed a drastic decrease in electron transfer activities around PSII, with little change in electron transfer activities around PSI, a phenomenon which was exaggerated under high light stress. The accumulation of plastoquinone-9 (PQ-9) was suppressed in the mutant, and >90% of the PQ-9 pool was reduced under growth light conditions. Non-photochemical quenching (NPQ) in the mutant functioned less efficiently, resulting from little contribution by energy-dependent quenching (qE). The ultrastructure of thylakoids in the mutant revealed that their grana were unstacked and transformed into loose and disordered structures. Light-harvesting complex (LHC)-containing large photosystem complexes and photosystem core complexes in the mutant were less abundant than those in wild-type plants. These results suggest that the lack of FBN5 causes a decrease in PQ-9 and imbalance of the redox state of PQ-9, resulting in misconducting both short-term and long-term control of the input of light energy to photosynthetic reaction centers. Furthermore, in the fbn5 mutant, the expression of genes involved in jasmonic acid biosynthesis was suppressed to ≤10% of that in the wild type under both growth-light and high-light conditions, suggesting that FBN5 functions as a transmitter of 1O2 in the stroma. Introduction In plants, light is not only a source of energy but also a source of information about their environment. The photosynthetic electron transfer driven by light can interact with molecular oxygen, leading to the formation of reactive oxygen species (ROS), mainly O2·– in PSI and mainly 1O2 in PSII (Asada 1999, Apel and Hirt 2004, Fischer et al. 2013). Production of ROS is significantly enhanced under photooxidative stress conditions where plants absorb excessive light relative to the photosynthetic activity. ROS attack various types of biological molecules such as DNA and proteins to damage cells/tissues, besides functioning as crucial cellular signals. In the photosynthetic apparatus, the synthesis of the PSII reaction center protein D1 is inhibited by ROS to impair photosynthetic function (Nishiyama et al. 2001, Vass 2012). Such impairment of PSII tends to remove the balance between both photosystems, falling into a vicious circle leading to further production of ROS. On the other hand, there is growing evidence that ROS can also serve as a protection mechanism against photoinhibition to prevent further damage of the system (Fischer et al. 2013, Foyer et al. 2017). To manage the resulting production of ROS, chloroplasts contain a variety of antioxidant mechanisms including soluble and lipophilic low molecular weight antioxidants, detoxification enzymes and repair mechanisms (Tarrago et al. 2008, Tripathi et al. 2009, Pospíšil 2014). For example, the plastoquinone (PQ) pool located on the acceptor side of PSII has been attributed a role in the regulation of gene expression and enzyme activities through its redox state (Pfannschmidt et al. 2001, Tikkanen et al. 2012, Rochaix 2013, Petrillo et al. 2014), as well as acting as an antioxidant in plant leaves to play a central photoprotective role (Ksas et al. 2015). In chloroplasts and chromoplasts, there are tiny granules, so-called plastoglobules (PGs) which function as a reservoir to accumulate lipids such as tocopherols and carotenoids that are antioxidants to quench ROS. PGs are lipoprotein bodies surrounded by a monolayer of phospholipid that is contiguous with the stroma-side leaflet of the thylakoid membrane (Austin et al. 2006), and contain structural proteins called fibrillins (FBNs) and enzymatic proteins as well as antioxidants (Brehelin et al. 2007). PGs vary in size and number during plastid development and differentiation, and are more abundant in response to environmental stresses [e.g. high light (HL), drought, high salinity and exposure to ozone] and during senescence (Rey et al. 2000, Simkin et al. 2007, Singh et al. 2010, Youssef et al. 2010, Ariizumi et al. 2014, Martinis et al. 2014). FBNs, which were first discovered in fibrils, the suborganellar structures in chromoplasts, are generally associated with PGs in leaf tissue and also termed plastid lipid-associated proteins (PAPs) or plastoglobulins (Pozueta-Romero et al. 1997, Kessler et al. 1999). FBNs are a large protein family present in photosynthetic organisms ranging from cyanobacteria to higher plants, and can be divided to 12 phylogenetic groups in which Groups 1, 3 and 7 have two ortholog proteins each (Singh and McNellis 2011). All FBNs possess chloroplast transit peptide sequences in their N-terminus. van Wijk’s group demonstrated, with proteome analyses of PGs and thylakoid membranes from Arabidopsis thaliana (Arabidopsis), that at least eight FBNs (FBN1a/1b/2/3a/4/7a/7b/8) are bound to PGs (Friso et al. 2004, Ytterberg et al. 2006). They further clarified the localization of FBNs in chloroplasts by mass spectrometry of isolated PGs and quantitative comparison with the proteomes of unfractionated leaves, thylakoids and stroma (Lundquist et al. 2012): the PG/thylakoid and PG/stroma abundance ratios of FBN1a were 34 and 176, respectively, which confirms that almost all of FBN1a is localized on PGs. From this study, FBN1a/1b/2/4/7a/7b/8 were localized in PGs and constituted 53% of the PG proteome mass, while FBN3a/3b/6/9 were localized in thylakoid membranes. FBN10 was present on both PGs and thylakoid membranes. In recent years, experimental research has made it clear that some FBNs, known as major structural proteins of PGs, are involved in the adaption to biotic/abiotic environmental stresses. FBN1a/1b/2-knockdown plants increased their sensitivity to photoinhibition of PSII, resulting in impairment of long-term acclimation to photooxidative stress imposed by HL combined with cold. Jasmonate biosynthesis related to light/cold stress is also regulated by the accumulation of PG-associated FBN1/2 proteins (Youssef et al. 2010). Gamez-Arjona et al. (2014) reported that FBN1a can interact with starch synthase 4 located in specific areas of the thylakoid membranes, suggesting that FBN1a functions in the regulation of synthesis of starch granules at specific regions of chloroplasts. FBN4 was also reported to be involved in the acquisition of resistance to the various stresses such as pathogenic infection and ozone exposure (Singh et al. 2010). FBN4-knockdown apple trees were more susceptible than their wild-type counterparts to the bacterial pathogen Erwinia amylovora and were more sensitive to ozone-induced tissue damage. PG osmiophilicity, which is considered to reflect the content of major antioxidants such as PQ, carotenoids and triacylglycerides, was decreased in FBN4-knockdown apple tree chloroplasts compared with the wild type. Indeed, the PQ level in mutant PGs was <10% of that in wild-type PGs (Singh et al. 2012). FBN4 is suggested to play a role in accumulating antioxidant lipids into PG, resulting in an increase in stress resistance. Little is known about the function and localization of FBN5 in stressed plants, compared with FBNs of major PG proteins. FBN5 belongs to Group 5, and is a unique minor protein of FBN family proteins because FBN5 is unlikely to be localized in PGs and thylakoid membranes, showing a low pI and the lowest hydrophobicity indices of the 16 FBN protein products (Lundquist et al. 2012). In our previous study, we initially identified FBN5 as one of the candidate genes functioning in freezing tolerance, using the activation-tagging callus lines generated by random insertion of strong enhancers (Otsubo et al. 2007). Recently, Kim et al. (2015) have demonstrated that FBN5 functions as a structural component involved in the biosynthesis of plastoquinone-9 (PQ-9). FBN5 binding to the hydrophobic solanesyl moiety, which is generated by solanesyl diphosphate (SPP) synthases (SPSs) 1 and 2, in the FBN5-B/SPS homodimeric complex stimulates their enzyme activities. They propose a model for PQ-9 biosynthesis in Arabidopsis whereby SPSs of the FBN5/dimeric SPS complex catalyze the sequential condensation of five molecules of isopentenyl diphosphate (IPP) with geranylgeranyl diphosphate (GGPP) to yield the all-trans form of SPP and facilitate both the conversion of SPP to PQ-9 and its translocation to the thylakoids and PGs. In this study, in order to clarify further an involvement of FBN5 in acclimation against abiotic environmental stresses, we have studied the structural and functional properties of FBN5 with fbn5-knockout and fbn5-complemented mutants. Results The T3 plants of the activation-tagged mutant containing the Cauliflower mosaic virus (CaMV) 35S enhancers, designated as the 18-16 line (Otsubo et al. 2007), were evaluated by freezing treatment at −14°C for 3 h (Fig. 1A). The calli from the 18-16 mutant line were more freezing tolerant than those from the wild-type plants. In this line, a single T-DNA activation-tag was inserted at position 3,058,129 on chromosome 5, by TAIL-PCR (thermal asymmetrical interlaced PCR) and Southern blotting (Fig. 1B). In four genes located in the vicinity of the enhancer insertion site, an At5g09820 gene, known as FBN5, was strongly expressed in the mutant line (Fig. 1C, D). Interestingly, there were two different sizes of transcripts of At5g09820 in reverse transcription–PCR (RT–PCR), of which the longer and shorter transcripts were tentatively named At5g09820a and At5g09820b, respectively. Fig. 1 View largeDownload slide Characterization of the activation-tagged line (18-16). (A) Evaluation of freezing tolerance with wild-type (WT) (left) and 18-16 (right) calli. After freezing at −14°C for 3 h, the calli were transferred to shoot-inducing medium (SIM). (B) Diagram showing the position of the T-DNA insertion (pPCVICEn4HPT) in the 18-16 line. HPT, hygromycin phosphotransferase; LB, left border; RB, right border; 4×En denotes four copies of 35S enhancer. (C, D) Expression profiles of genes located in the vicinity of the T-DNA insertion site in wild-type and 18-16 seedlings which were kept at 23 or 2°C for 5 d, analyzed by semi-quantitative RT–PCR using F2/R4 primers (C) and quantitative RT–PCR using F2/R3 or F2/R2 primers (D), respectively. Fig. 1 View largeDownload slide Characterization of the activation-tagged line (18-16). (A) Evaluation of freezing tolerance with wild-type (WT) (left) and 18-16 (right) calli. After freezing at −14°C for 3 h, the calli were transferred to shoot-inducing medium (SIM). (B) Diagram showing the position of the T-DNA insertion (pPCVICEn4HPT) in the 18-16 line. HPT, hygromycin phosphotransferase; LB, left border; RB, right border; 4×En denotes four copies of 35S enhancer. (C, D) Expression profiles of genes located in the vicinity of the T-DNA insertion site in wild-type and 18-16 seedlings which were kept at 23 or 2°C for 5 d, analyzed by semi-quantitative RT–PCR using F2/R4 primers (C) and quantitative RT–PCR using F2/R3 or F2/R2 primers (D), respectively. Cloning cDNAs of these transcripts, we found that they are two types of At5g08920 splicing variants, as shown in Fig. 2A: the transcript At5g09820a encodes 259 amino acids because of the existence of a stop codon in intron 5, while the transcript At5g09820b encodes the 273 amino acids of the full-length FBN5. Thus, in this study, At5g09820a and At5g09820b were conveniently named FBN5-259 and FBN5(-273), respectively. These FBN5 mRNA splicing variants were also reported by Kim et al. (2015). The expression of FBN5 under normal growth conditions was much greater than that of FBN5-259, while under cold conditions the latter became comparable with the former (Fig. 2B). To clarify the function of FBN5, we have investigated the Salk T-DNA insertion line (SALK_064597) in which FBN5 is functionally deleted. Two copies of T-DNA were inserted in tandem at position 3,057,168 of chromosome 5 in the first exon of FBN5, resulting in impairment of FBN5 transcription (Fig. 2C;Supplementary Fig. S1). A homozygous mutant of SALK_064597, which we named the fbn5 mutant, has been reported to be seedling lethal (Savage et al. 2013, Kim et al. 2015). We reconfirmed that continuous light conditions at 80 µmol m–2 s–1 caused all fbn5 plants to wilt and die. However, when the fbn5 plants were transferred to a 12 h:12 h day/night photoperiod at 80 µmol m–2s–1, approximately 20% of the plants did not die but were able to produce seeds 11–12 weeks after germination; they showed a dwarf phenotype (Supplementary Fig. S2). The fbn5 mutant was genotyped by PCR analysis using primers specific for the T-DNA and FBN5 (Supplementary Fig. S1; Table S1). A 550 bp band was detected with the F1/RBa2 but not with the F1/R1 primers, confirming that the mutant employed for this study is homozygous for the T-DNA insertion. The linkages of identical T-DNAs were reported to be frequently integrated at the same locus, independent of the transformation method or species used (De Neve et al. 1997). We have developed transgenic Arabidopsis lines, designated as fbn5/FBN5 and fbn5/FBN-259, overexpressing FBN5 and FBN5-259 cDNAs, respectively, driven under the control of the CaMV 35S promoter. The fbn5/FBN5 and fbn5/FBN5-259 lines exhibited mRNA levels increased by approximately 6- and 10-fold, respectively, compared with wild-type plants (Fig. 2C). Fig. 2 View largeDownload slide Alternative splicing of a FBN5 gene, and the organ-specific expression of splicing variants in wild-type plants. (A) Structures of FBN5 genomic DNA and two splicing variants. Roman numerals denote intron numbers. (B) Relative expression levels of FBN5-259 (white bar) and FBN5 (black bar). The tissues shown here were obtained from non-acclimated (left) and cold-acclimated (right) wild-type plants. The expression level of FBN5-259 in non-acclimated young leaves was set to 1. (C) Relative expression levels of FBN5 and FBN5-259 in wild-type, fbn5 (SALK_064597) and fbn5-complemented (fbn5/FBN5-259 and fbn5/FBN5) plants. Data are presented as the means ± SE (n = 4). Real-time RT–PCR primers for FBN5 (F2/R2) and FBN5-259 (F2/R3) were indicated in (A) and Supplementary Table S1. Fig. 2 View largeDownload slide Alternative splicing of a FBN5 gene, and the organ-specific expression of splicing variants in wild-type plants. (A) Structures of FBN5 genomic DNA and two splicing variants. Roman numerals denote intron numbers. (B) Relative expression levels of FBN5-259 (white bar) and FBN5 (black bar). The tissues shown here were obtained from non-acclimated (left) and cold-acclimated (right) wild-type plants. The expression level of FBN5-259 in non-acclimated young leaves was set to 1. (C) Relative expression levels of FBN5 and FBN5-259 in wild-type, fbn5 (SALK_064597) and fbn5-complemented (fbn5/FBN5-259 and fbn5/FBN5) plants. Data are presented as the means ± SE (n = 4). Real-time RT–PCR primers for FBN5 (F2/R2) and FBN5-259 (F2/R3) were indicated in (A) and Supplementary Table S1. Since the fbn5 plants transformed with cDNAs of FBN5 and FBN5-259 were phenotypically similar to wild-type and fbn5 plants, respectively, FBN5 was found to be a functional transcript (Supplementary Fig. S2). The structure of leaf tissues of 6-week-old wild-type, fbn5 and fbn5/FBN5 plants was examined with a light microscope (Fig. 3A–F). Compared with the wild-type plants, the fbn5 plants possessed cells with smaller size and thinner leaves, and fbn5/FBN5 plants exhibited thicker leaves. The mean number of chloroplasts per palisade mesophyll cell was 5.7 in fbn5 plants, while those in wild-type and fbn5/FBN5 plants were 10.1 and 9.5, respectively. Under the HL (800 µmol m-2 s-1) condition, starch accumulation was well observed in the chloroplast in wild-type and fbn5/FBN5 plants, but not in fbn5 plants (Fig. 3D–F). From TEM images shown in Fig. 3G–L, the fbn5 plants were found to possess abnormalities in chloroplast and thylakoid morphology, including swollen thylakoid vesicles in a granum, disorganized grana stacks, alterations in the relative proportions of grana and stroma thylakoids, and decreased starch granules. The chloroplast ultrastructure in the fbn5/FBN5 plants was similar to that of the wild-type plants, except for producing more abundant starch granules. Such architectural change in thylakoid membranes might affect the assembly of PSI/II reaction center complexes. Indeed, we found, with the native green gel analysis according to Allen and Staehelin (1991), that the amounts of light-harvesting complex (LHC)-containing large photosystem complexes (RC-LHC), photosystem complexes (RC-core) and LHCII trimers (LHCII-A, B, C and D) in fbn5 plants were much lower than those in wild-type and fbn5/FBN5 plants (Fig. 4). Since LHCII trimers are responsible for membrane stacking (Wan et al. 2014), a decrease in LHCII trimers may lead to the lack of grana stacking. Fig. 3 View largeDownload slide Light and ultramicroscopic photomicrographs of leaf tissue and chloroplast structures from wild-type, fbn5 and fbn5/FBN5 plants. (A–F) Cross-sections of rosette leaves of wild-type (A, D), fbn5 (B, E), and fbn5/FBN5 (C, F) plants. Upper and lower panels show the cross-section of leaves before and after 4 h HL treatment, respectively. Leaf pieces fixed and embedded for electron micrograph analysis were sliced 1 µm thick and stained with 0.5% toluidine blue. Scale bar = 50 μm. (G–L) Transmission electron micrograph of chloroplasts in wild-type (G, J), fbn5 (H, K) and fbn5/FBN5 (I, L) plants. Upper and lower panels show chloroplasts before and after 4 h HL treatment, respectively. Scale bar = 1 μm. Fig. 3 View largeDownload slide Light and ultramicroscopic photomicrographs of leaf tissue and chloroplast structures from wild-type, fbn5 and fbn5/FBN5 plants. (A–F) Cross-sections of rosette leaves of wild-type (A, D), fbn5 (B, E), and fbn5/FBN5 (C, F) plants. Upper and lower panels show the cross-section of leaves before and after 4 h HL treatment, respectively. Leaf pieces fixed and embedded for electron micrograph analysis were sliced 1 µm thick and stained with 0.5% toluidine blue. Scale bar = 50 μm. (G–L) Transmission electron micrograph of chloroplasts in wild-type (G, J), fbn5 (H, K) and fbn5/FBN5 (I, L) plants. Upper and lower panels show chloroplasts before and after 4 h HL treatment, respectively. Scale bar = 1 μm. Fig. 4 View largeDownload slide Native green gel electrophoresis of Chl–protein complexes isolated from wild-type, fbn5 and fbn5/FBN5 plants. Thylakoid membranes were isolated from 6-week-old plants, which had been grown under 12 h light/12 h dark or continuous light conditions for 2 weeks before the experiment. Separated bands are assigned according to Allen and Staehelin (1991) as follows: RC-LHC, large PSI and PSII complexes both with attached antenna; RC-Core, several partial PSI and PSII complexes that have been largely stripped of antennae; LHCII trimer, the trimeric form of the PSII antenna LHCII; SC, small complexes containing Chls. Fig. 4 View largeDownload slide Native green gel electrophoresis of Chl–protein complexes isolated from wild-type, fbn5 and fbn5/FBN5 plants. Thylakoid membranes were isolated from 6-week-old plants, which had been grown under 12 h light/12 h dark or continuous light conditions for 2 weeks before the experiment. Separated bands are assigned according to Allen and Staehelin (1991) as follows: RC-LHC, large PSI and PSII complexes both with attached antenna; RC-Core, several partial PSI and PSII complexes that have been largely stripped of antennae; LHCII trimer, the trimeric form of the PSII antenna LHCII; SC, small complexes containing Chls. The fbn5 plants were susceptible to long-day treatment such as a photoperiodic program of 16 h light:8 h dark or 24 h light (continuous light), and were withered. To evaluate how FBN5 affects photosynthetic capability, we have studied the effects of strong light on the maximum quantum efficiency of PSII photochemistry (Fv/Fm) in the wild-type and mutant plants (Fig. 5A). The HL treatment hardly decreased Fv/Fm values of the wild-type and fbn5/FBN5 plants during the time periods examined. On the other hand, Fv/Fm values of the fbn5 plants were decreased from the initial value of 0.6 to 0.02 by HL: longer exposure to HL rendered the fbn5 plants incapable of recovery of Fv/Fm under the subsequent growth light (GL, 80 µmol m–2 s–1) conditions (open symbols in Fig. 5A). To evaluate whether photodamage is enhanced and/or the repair is suppressed in fbn5 plants, we have studied the rates of photodamage to PSII by comparing photoinactivation kinetics in the presence of an inhibitor of protein synthesis, lincomycin, with the wild-type (Col-0) and fbn5 plants (Supplementary Fig. S3). Photoinactivation obeyed pseudo-first-order kinetics with respect to illumination time in both plants. The rate constant for photoinactivation, 0.073 h–1, at a light intensity of 80 µmol m–2 s–1 in fbn5 plants was much greater than that in the wild-type plants (0.0125 h–1). The addition of lincomycin increased rate constants to the same degree in both plants. These results have revealed that photodamage is enhanced in the fbn5 plants with little suppression of repair. We have also investigated the electron transfer activities around both photosystems, with thylakoid membranes isolated from wild-type and mutant leaves (Fig. 5B). Electron transfer activities around PSII in the fbn5 plants were drastically decreased by the HL exposure, while those for the wild-type and fbn5/FBN5 plants were not affected. Meanwhile, electron transfer activities of PSI remained intact after the HL treatment in all types of plants examined in this study. Taken together, our data provide evidence that dysfunction of FBN5 specifically induces photodamage of PSII. Fig. 5 View largeDownload slide Effects of HL on photosynthetic electron transfer in wild-type, fbn5 and fbn5/FBN5 plants. (A) The maximum quantum efficiency of PSII (Fv/Fm) under HL conditions. Filled circles, squares and triangles show the values for Fv/Fm of 6-week-old wild-type, fbn5/FBN5 and fbn5 plants, respectively, which were exposed to HL for a given time. Open symbols show the Fv/Fm values obtained with fbn5 plants which were transferred to GL conditions at a given time after HL exposure for 1, 3 or 6 h. Data are mean values of six separate experiments ± SD. (B) PSII/ PSI electron transfer activities before (gray) or after (white) 4 h HL treatment. Data are normalized to the values obtained with wild-type (WT) plants without any HL exposure. The rates of O2 evolution and consumption of 81 and 180 µmol O2 mg Chl–1 h–1, respectively, in wild-type plants were set at 100%. Data are presented as the means ± SD (n = 3–4). The asterisk marks statistically significant differences (P < 0.05 by Student’s t-test) relative to samples without any HL treatment. Fig. 5 View largeDownload slide Effects of HL on photosynthetic electron transfer in wild-type, fbn5 and fbn5/FBN5 plants. (A) The maximum quantum efficiency of PSII (Fv/Fm) under HL conditions. Filled circles, squares and triangles show the values for Fv/Fm of 6-week-old wild-type, fbn5/FBN5 and fbn5 plants, respectively, which were exposed to HL for a given time. Open symbols show the Fv/Fm values obtained with fbn5 plants which were transferred to GL conditions at a given time after HL exposure for 1, 3 or 6 h. Data are mean values of six separate experiments ± SD. (B) PSII/ PSI electron transfer activities before (gray) or after (white) 4 h HL treatment. Data are normalized to the values obtained with wild-type (WT) plants without any HL exposure. The rates of O2 evolution and consumption of 81 and 180 µmol O2 mg Chl–1 h–1, respectively, in wild-type plants were set at 100%. Data are presented as the means ± SD (n = 3–4). The asterisk marks statistically significant differences (P < 0.05 by Student’s t-test) relative to samples without any HL treatment. To evaluate whether the fbn5 plants lack the capability for protecting the photosynthetic machinery against overexcitation and subsequent damage, we have studied parameters for non-photochemical (NPQ, qE and qI) and photochemical (qP) quenching, and the quantum yield of the Chl photophysical decay pathways (ΦC) in wild-type and fbn5 plants grown under GL conditions (Table 1). NPQ is a parameter for non-photochemical Chl fluorescence quenching, monitoring the apparent rate constant for non-radiative decay (heat loss) from PSII and its antennae. qE and qI, which are major components of NPQ, are related to ΔpH-triggered conformation change in the photosynthetic membranes and photoinhibition/damage to PSII reaction centers, respectively (Ruban 2016). In wild-type plants, at increasing actinic light intensities, NPQ increased drastically, concomitantly with an increase in both qE and qI. In fbn5 plants, NPQ increased from 0.08 to 0.36 with increasing light intensities, to which qI, but not qE, was a major contributor. The fluorescence traces obtained in fbn5 plants, in which the quenching of Chl fluorescence was largely suppressed, were very similar to that in npq4 mutant plants lacking PsbS (Roach and Krieger-Liszkay 2012). These results have suggested that the fbn5 plants lose the capacity for ΔpH-triggered energy dissipation events such as protonation of antenna components, the xanthophyll cycle(s), LHCII rearrangement/aggregation and the formation of the NPQ quencher. Table 1 Various PAM parameters in wild-type and fbn5 plants Light intensity of excitation light (µmol m–2s–1) PAM parameters NPQ qE qI qP ΦC WT 20 0.23 ± 0.07 0.09 ± 0.06 0.14 ± 0.01 0.91 ± 0.04 0.23 ± 0.01 80 0.27 ± 0.02 0.06 ± 0.03 0.20 ± 0.04 0.86 ± 0.01 0.26 ± 0.01 400 1.11 ± 0.14 0.88 ± 0.09 0.24 ± 0.08 0.47 ± 0.01 0.33 ± 0.02 fbn5 20 0.08 ± 0.02 − 0.08 ± 0.03 0.26 ± 0.09 0.78 ± 0.06 80 0.14 ± 0.01 0.015 ± 0.01 0.12 ± 0.02 0.16 ± 0.07 0.79 ± 0.05 400 0.36 ± 0.05 0.032 ± 0.02 0.32 ± 0.06 0.06 ± 0.02 0.71 ± 0.03 Light intensity of excitation light (µmol m–2s–1) PAM parameters NPQ qE qI qP ΦC WT 20 0.23 ± 0.07 0.09 ± 0.06 0.14 ± 0.01 0.91 ± 0.04 0.23 ± 0.01 80 0.27 ± 0.02 0.06 ± 0.03 0.20 ± 0.04 0.86 ± 0.01 0.26 ± 0.01 400 1.11 ± 0.14 0.88 ± 0.09 0.24 ± 0.08 0.47 ± 0.01 0.33 ± 0.02 fbn5 20 0.08 ± 0.02 − 0.08 ± 0.03 0.26 ± 0.09 0.78 ± 0.06 80 0.14 ± 0.01 0.015 ± 0.01 0.12 ± 0.02 0.16 ± 0.07 0.79 ± 0.05 400 0.36 ± 0.05 0.032 ± 0.02 0.32 ± 0.06 0.06 ± 0.02 0.71 ± 0.03 Calculation formulae of PAM parameters are described in the Materials and Methods. Data are means ± SD (n = 3). Table 1 Various PAM parameters in wild-type and fbn5 plants Light intensity of excitation light (µmol m–2s–1) PAM parameters NPQ qE qI qP ΦC WT 20 0.23 ± 0.07 0.09 ± 0.06 0.14 ± 0.01 0.91 ± 0.04 0.23 ± 0.01 80 0.27 ± 0.02 0.06 ± 0.03 0.20 ± 0.04 0.86 ± 0.01 0.26 ± 0.01 400 1.11 ± 0.14 0.88 ± 0.09 0.24 ± 0.08 0.47 ± 0.01 0.33 ± 0.02 fbn5 20 0.08 ± 0.02 − 0.08 ± 0.03 0.26 ± 0.09 0.78 ± 0.06 80 0.14 ± 0.01 0.015 ± 0.01 0.12 ± 0.02 0.16 ± 0.07 0.79 ± 0.05 400 0.36 ± 0.05 0.032 ± 0.02 0.32 ± 0.06 0.06 ± 0.02 0.71 ± 0.03 Light intensity of excitation light (µmol m–2s–1) PAM parameters NPQ qE qI qP ΦC WT 20 0.23 ± 0.07 0.09 ± 0.06 0.14 ± 0.01 0.91 ± 0.04 0.23 ± 0.01 80 0.27 ± 0.02 0.06 ± 0.03 0.20 ± 0.04 0.86 ± 0.01 0.26 ± 0.01 400 1.11 ± 0.14 0.88 ± 0.09 0.24 ± 0.08 0.47 ± 0.01 0.33 ± 0.02 fbn5 20 0.08 ± 0.02 − 0.08 ± 0.03 0.26 ± 0.09 0.78 ± 0.06 80 0.14 ± 0.01 0.015 ± 0.01 0.12 ± 0.02 0.16 ± 0.07 0.79 ± 0.05 400 0.36 ± 0.05 0.032 ± 0.02 0.32 ± 0.06 0.06 ± 0.02 0.71 ± 0.03 Calculation formulae of PAM parameters are described in the Materials and Methods. Data are means ± SD (n = 3). A parameter estimating the fraction of PSII centers in the open state (with QA oxidized representing the redox state of the PQ pool), qP, in wild-type plants was decreased from 0.91 to 0.47 with increasing actinic light intensities. The fbn5 plants were shown to have significantly lower qP; it was 0.26 even at a low actinic light intensity of 20 µmol m–2 s–1 and further decreased to 0.06 at the relatively high actinic light intensity of 400 µmol m–2 s–1. These results indicate that PSII centers in the fbn5 plants are almost closed, or in the reduced state of the PQ pool, even under GL conditions. Furthermore, the values of the parameter ΦC, estimating 1O2 generation by PSII (Ahn et al. 2009; Kasajima et al. 2009), were found to be much greater in fbn5 plants than in wild-type plants at the light intensities examined, suggesting that fbn5 plants generate a considerable amount of 1O2 even under weak light. We next investigated the effects of HL on xanthophylls operating in non-photochemical quenching (Table 2). In HL, violaxanthin (V) is converted into zeaxanthin (Z), via the intermediate antheraxanthin (A), by the enzyme violaxanthin de-epoxidase, resulting in the binding of zeaxanthin to light-harvesting proteins and the subsequent quenching and heat dissipation (Demmig-Adams and Adams 1992, Horton et al. 1996, Jahns and Holzwarth 2012). In wild-type and fbn5/FBN5 plants, the content of zeaxanthin and antheraxanthin was increased by 3.4- and 3.2-fold, respectively, by the HL treatment, and the de-epoxidation status of the xanthophyll cycle pigments [i.e. (A + Z)/(V + A + Z)] was increased. This indicates that a xanthophyll cycle functions normally under HL conditions in wild-type and fbn5/FBN5 plants. In the fbn5 plants, the contents of zeaxanthin and antheraxanthin were slightly greater compared with those in the wild-type plants under GL conditions, while they were increased by 1.4-fold by the HL treatment, but the de-epoxidation status was not increased substantially. The fbn5 plants showed a lower Chl content per unit of leaf area (Supplementary Fig. S4). These results suggest that in the fbn5 plants photoinhibition occurs even under GL conditions and that a xanthophyll cycle inefficiently functions in non-photochemical quenching processes. Table 2 Pigment contents in leaves of wild-type, fbn5 and fbn5/FBN5 plants Pigment content (nmol g–1 FW) Neo Vio Ant Zea Lut β-car Chl a + b Chl a/b (A + Z)/ (V + A + Z) GL WT 47.8 ± 5.80 62.6 ± 9.26 2.79 ± 0.45 3.98 ± 1.56 231 ± 26.1 96.4 ± 6.72 1,742 ± 156 3.31 ± 0.03 0.10 ± 0.03 fbn5 75.1 ± 3.55 124 ± 7.04 8.88 ± 4.10 13.3 ± 5.80 329 ± 45.3 129 ± 10.2 2,690 ± 454 2.64 ± 0.16 0.15 ± 0.03 fbn5/FBN5 65.0 ± 5.2 72.5 ± 3.32 3.35 ± 0.41 7.4 ± 2.20 305 ± 16.2 113 ± 4.9 2,069 ± 166 3.16 ± 0.04 0.13 ± 0.03 HL WT 64.1 ± 12.9 48.2 ± 10.3 13.2 ± 5.16 9.84 ± 7.20 328 ± 63.8 112 ± 7.66 1,909 ± 181 3.10 ± 0.05 0.33 ± 0.07 fbn5 72.2 ± 1.22 74.4 ± 0.82 22.3 ± 3.02 7.67 ± 3.80 356 ± 14.9 97.5 ± 12.4 2,402 ± 336 2.67 ± 0.15 0.28 ± 0.04 fbn5/FBN5 75.8 ± 1.8 46.8 ± 3.71 13.35 ± 2.87 21.5 ± 13.6 389 ± 67.0 152 ± 2.8 2,492 ± 235 3.13 ± 0.04 0.42 ± 0.07 Pigment content (nmol g–1 FW) Neo Vio Ant Zea Lut β-car Chl a + b Chl a/b (A + Z)/ (V + A + Z) GL WT 47.8 ± 5.80 62.6 ± 9.26 2.79 ± 0.45 3.98 ± 1.56 231 ± 26.1 96.4 ± 6.72 1,742 ± 156 3.31 ± 0.03 0.10 ± 0.03 fbn5 75.1 ± 3.55 124 ± 7.04 8.88 ± 4.10 13.3 ± 5.80 329 ± 45.3 129 ± 10.2 2,690 ± 454 2.64 ± 0.16 0.15 ± 0.03 fbn5/FBN5 65.0 ± 5.2 72.5 ± 3.32 3.35 ± 0.41 7.4 ± 2.20 305 ± 16.2 113 ± 4.9 2,069 ± 166 3.16 ± 0.04 0.13 ± 0.03 HL WT 64.1 ± 12.9 48.2 ± 10.3 13.2 ± 5.16 9.84 ± 7.20 328 ± 63.8 112 ± 7.66 1,909 ± 181 3.10 ± 0.05 0.33 ± 0.07 fbn5 72.2 ± 1.22 74.4 ± 0.82 22.3 ± 3.02 7.67 ± 3.80 356 ± 14.9 97.5 ± 12.4 2,402 ± 336 2.67 ± 0.15 0.28 ± 0.04 fbn5/FBN5 75.8 ± 1.8 46.8 ± 3.71 13.35 ± 2.87 21.5 ± 13.6 389 ± 67.0 152 ± 2.8 2,492 ± 235 3.13 ± 0.04 0.42 ± 0.07 Pigment measurements were performed before and after exposure of GL (80 µmol m–2 s-1)-grown plants to HL (800 µmol m–2 s–1) for 4 h. Data are presented as the means ± SD (n = 4–5). Neo, neoxanthin; Vio, violaxanthin (V); Ant, antheraxanthin (A); Zea, zeaxanthin (Z); Lut, lutein; β-car, β-carotene. Table 2 Pigment contents in leaves of wild-type, fbn5 and fbn5/FBN5 plants Pigment content (nmol g–1 FW) Neo Vio Ant Zea Lut β-car Chl a + b Chl a/b (A + Z)/ (V + A + Z) GL WT 47.8 ± 5.80 62.6 ± 9.26 2.79 ± 0.45 3.98 ± 1.56 231 ± 26.1 96.4 ± 6.72 1,742 ± 156 3.31 ± 0.03 0.10 ± 0.03 fbn5 75.1 ± 3.55 124 ± 7.04 8.88 ± 4.10 13.3 ± 5.80 329 ± 45.3 129 ± 10.2 2,690 ± 454 2.64 ± 0.16 0.15 ± 0.03 fbn5/FBN5 65.0 ± 5.2 72.5 ± 3.32 3.35 ± 0.41 7.4 ± 2.20 305 ± 16.2 113 ± 4.9 2,069 ± 166 3.16 ± 0.04 0.13 ± 0.03 HL WT 64.1 ± 12.9 48.2 ± 10.3 13.2 ± 5.16 9.84 ± 7.20 328 ± 63.8 112 ± 7.66 1,909 ± 181 3.10 ± 0.05 0.33 ± 0.07 fbn5 72.2 ± 1.22 74.4 ± 0.82 22.3 ± 3.02 7.67 ± 3.80 356 ± 14.9 97.5 ± 12.4 2,402 ± 336 2.67 ± 0.15 0.28 ± 0.04 fbn5/FBN5 75.8 ± 1.8 46.8 ± 3.71 13.35 ± 2.87 21.5 ± 13.6 389 ± 67.0 152 ± 2.8 2,492 ± 235 3.13 ± 0.04 0.42 ± 0.07 Pigment content (nmol g–1 FW) Neo Vio Ant Zea Lut β-car Chl a + b Chl a/b (A + Z)/ (V + A + Z) GL WT 47.8 ± 5.80 62.6 ± 9.26 2.79 ± 0.45 3.98 ± 1.56 231 ± 26.1 96.4 ± 6.72 1,742 ± 156 3.31 ± 0.03 0.10 ± 0.03 fbn5 75.1 ± 3.55 124 ± 7.04 8.88 ± 4.10 13.3 ± 5.80 329 ± 45.3 129 ± 10.2 2,690 ± 454 2.64 ± 0.16 0.15 ± 0.03 fbn5/FBN5 65.0 ± 5.2 72.5 ± 3.32 3.35 ± 0.41 7.4 ± 2.20 305 ± 16.2 113 ± 4.9 2,069 ± 166 3.16 ± 0.04 0.13 ± 0.03 HL WT 64.1 ± 12.9 48.2 ± 10.3 13.2 ± 5.16 9.84 ± 7.20 328 ± 63.8 112 ± 7.66 1,909 ± 181 3.10 ± 0.05 0.33 ± 0.07 fbn5 72.2 ± 1.22 74.4 ± 0.82 22.3 ± 3.02 7.67 ± 3.80 356 ± 14.9 97.5 ± 12.4 2,402 ± 336 2.67 ± 0.15 0.28 ± 0.04 fbn5/FBN5 75.8 ± 1.8 46.8 ± 3.71 13.35 ± 2.87 21.5 ± 13.6 389 ± 67.0 152 ± 2.8 2,492 ± 235 3.13 ± 0.04 0.42 ± 0.07 Pigment measurements were performed before and after exposure of GL (80 µmol m–2 s-1)-grown plants to HL (800 µmol m–2 s–1) for 4 h. Data are presented as the means ± SD (n = 4–5). Neo, neoxanthin; Vio, violaxanthin (V); Ant, antheraxanthin (A); Zea, zeaxanthin (Z); Lut, lutein; β-car, β-carotene. Based on a quantitative analysis of α-tocopherol and PQ-9, the amount of α-tocopherol in chloroplasts isolated from fbn5 plants was twice as high as in wild-type plants, while the amount of PQ-9 in chloroplasts isolated from fbn5 plants was 5-fold lower in wild-type plants (Fig. 6). The amounts of these lipids were gradually decreased during HL treatment in the wild-type and fbn5 plants. The ratio of reduced PQ-9 to total PQ-9 was increased from 50% to 60% by the 4 h HL treatment in wild-type plants, whereas it remained significantly high ( >90%) in fbn5 plants. Fig. 6 View largeDownload slide Quantitative determination of α-tocopherol, PQ-9 and plastoquinol-9. Lipids were extracted from leaves of 5- to 6-week-old plants during 4 h HL treatment, and analyzed with HPLC as described in the Materials and Methods. Data are presented as the means ± SD (n =3). Fig. 6 View largeDownload slide Quantitative determination of α-tocopherol, PQ-9 and plastoquinol-9. Lipids were extracted from leaves of 5- to 6-week-old plants during 4 h HL treatment, and analyzed with HPLC as described in the Materials and Methods. Data are presented as the means ± SD (n =3). FBN5 possessing a chloroplast transit signal at the N-terminus was previously reported to be present in the stroma, by mass spectroscopy analyses of the total leaf, thylakoid and stroma (Lundquist et al. 2012), and immunoblots with an anti-green fluorescent protein (GFP) antibody in transformed leaf protoplasts expressing the FBN5–GFP fusion proteins (Kim et al. 2015). We pursued the clarification of the subcellular localization and accumulation of FBN5 by quantitative immunoblot analysis of simply fractionated chloroplasts isolated from 6-week-old leaves with polyclonal antibodies to epitopes in the C-terminus of FBN5. Fig. 7 clearly shows the band with the predicted molecular weight (23.9 kDa) of FBN5 in only the stromal fraction; in the thylakoid fraction, no band with a molecular weight similar to FBN5 was detected. This result indicates that a FBN5 mature protein is localized in the stroma. When the plants were subjected to HL or low temperature, significant accumulation of FBN5 in wild-type plants was observed (Fig. 7B, C). The exposure of the plants to HL for 4 h did not induce distinct accumulation of FBN5, but 24 h HL exposure stimulated the accumulation of FBN5 by about 50% as compared with GL exposure. The exposure of the plants to low temperature (8°C) also enhanced the accumulation of FBN5, which was diminished by the combination of HL and low temperature (Fig. 7C). The accumulation of FBN-1a/b, -7b and -8 in PGs was reported to be increased by the HL treatment (Ytterberg et al. 2006). These results indicate that the HL or cold treatment modulates the rates of the synthesis and degradation of FBN5 in a different manner. Fig. 7 View largeDownload slide Immunodetection of FBN5 in stroma and thylakoid fractions. (A) Intact chloroplasts from wild-type, fbn5 and fbn5/FBN5 plants were separated into stromal and thylakoid fractions; equivalent amounts (10 µg of protein) were subject to SDS–PAGE, and then Western blotting using antibodies against FBN5, D1 (as a marker for the thylakoid membrane) and RbcL (as a stromal marker). C, intact chloroplasts; T, thylakoids; S, stroma. (B, C) Immunoblot analysis and ImageJ densitometry analysis of FBN5 in the crude stroma. Chloroplasts were prepared from rosette leaves from wild-type plants exposed to HL for 4 or 24 h at 4 or 23°C. Data are normalized to the values obtained under GL conditions at 23°C. Data are presented as the means ± SD (n = 3). Asterisks indicate a statistically significant difference relative to the values obtained under GL conditions at 23°C (P < 0.05 by Student’s t-test). Experiments in (B) and (C) were done separately with different preparations. Fig. 7 View largeDownload slide Immunodetection of FBN5 in stroma and thylakoid fractions. (A) Intact chloroplasts from wild-type, fbn5 and fbn5/FBN5 plants were separated into stromal and thylakoid fractions; equivalent amounts (10 µg of protein) were subject to SDS–PAGE, and then Western blotting using antibodies against FBN5, D1 (as a marker for the thylakoid membrane) and RbcL (as a stromal marker). C, intact chloroplasts; T, thylakoids; S, stroma. (B, C) Immunoblot analysis and ImageJ densitometry analysis of FBN5 in the crude stroma. Chloroplasts were prepared from rosette leaves from wild-type plants exposed to HL for 4 or 24 h at 4 or 23°C. Data are normalized to the values obtained under GL conditions at 23°C. Data are presented as the means ± SD (n = 3). Asterisks indicate a statistically significant difference relative to the values obtained under GL conditions at 23°C (P < 0.05 by Student’s t-test). Experiments in (B) and (C) were done separately with different preparations. To evaluate how FBN5 functions in the processes of acclimation, we have studied the effects of FBN5 on the expression of FBN family genes and stress-related genes (Fig. 8). These genes were selected from the FBN family and major genes of the following pathways which were previously reported to have more involvement with the FBN family; photosynthesis, PQ-9 and α-tocopherol biosynthesis, the xanthophyll cycle and carotenoid biosynthesis, ABA biosynthesis and jasmonate biosynthesis (Fig. 8A–F). In wild-type plants, the expression of FBN5 was increased by 4-fold in the 1 h HL treatment, but was subsequently decreased gradually by further HL treatment. FBN-1a, -1b, -2 and -4 are reported probably to be required for resistance to light/cold stress. Expression of FBN1a and FBN1b was significantly increased under HL in wild-type plants, while the expression of FBN2 and FBN4 was transiently increased at the beginning of HL treatment. The fbn5 plants showed the suppression of gene expression of FBN-1a, -1b and -2 during HL treatment, with some differences in the extent of their expression (Fig. 8A). These results suggest that the functional link between FBN5 and major members of the FBN family exists. Fig. 8 View largeDownload slide Effects of HL on gene expression of FBN family, photosynthesis- and stress-related genes. Transcript levels of the following genes in mature leaves of 6-week-old wild-type (circle) and fbn5 (triangle) plants were measured during 4 h HL treatment: (A) FBN5, FBN1a, FBN1b, FBN2 and FBN4. (B) PsbA, PSII subunit A (D1); PsbB, PSII subunit B (CP47); LHCB4.2, PSII light-harvesting complex gene 4.2; STN7/8, state transition 7/8. (C) SPS1/2, solanesyl diphosphate synthase 1/2; HST, homogentisate solanesyltransferase; HPT, homogentisate phytyltransferase. (D) PsbS, PSII subunit S; ZDS, ζ-carotene desaturase; VDE, violaxanthin de-epoxidase; ZEP, zeaxanthin epoxidase. (E) NCED3, 9-cis-epoxycarotenoid dioxygenase 3. (F) LOX2, lipoxygenase 2; AOS, allene oxide synthase; AOC2, allene oxide cyclase 2. mRNA levels were determined by quantitative real-time PCR using gene-specific primers (Supplementary Table S1), and were shown relative to the expression of ACT2 in each sample. Data are presented as the means ± SE (n =3–4). The genes up- and down-regulated by the HL treatment in fbn5 plants compared with the wild type are highlighted in yellow and blue, respectively. Fig. 8 View largeDownload slide Effects of HL on gene expression of FBN family, photosynthesis- and stress-related genes. Transcript levels of the following genes in mature leaves of 6-week-old wild-type (circle) and fbn5 (triangle) plants were measured during 4 h HL treatment: (A) FBN5, FBN1a, FBN1b, FBN2 and FBN4. (B) PsbA, PSII subunit A (D1); PsbB, PSII subunit B (CP47); LHCB4.2, PSII light-harvesting complex gene 4.2; STN7/8, state transition 7/8. (C) SPS1/2, solanesyl diphosphate synthase 1/2; HST, homogentisate solanesyltransferase; HPT, homogentisate phytyltransferase. (D) PsbS, PSII subunit S; ZDS, ζ-carotene desaturase; VDE, violaxanthin de-epoxidase; ZEP, zeaxanthin epoxidase. (E) NCED3, 9-cis-epoxycarotenoid dioxygenase 3. (F) LOX2, lipoxygenase 2; AOS, allene oxide synthase; AOC2, allene oxide cyclase 2. mRNA levels were determined by quantitative real-time PCR using gene-specific primers (Supplementary Table S1), and were shown relative to the expression of ACT2 in each sample. Data are presented as the means ± SE (n =3–4). The genes up- and down-regulated by the HL treatment in fbn5 plants compared with the wild type are highlighted in yellow and blue, respectively. The expression patterns of photosynthesis-related genes under HL stress were similar in wild-type and fbn5 plants: the peaks of expression of PsbA, PsbB and LHCB4.2 genes appeared at early stages of HL treatment, whereas STN7 and STN8 whose products phosphorylate LHCII and PSII core proteins, respectively, were almost constantly expressed (Fig. 8B). In particular, the lack of FBN5 suppressed the expression of PsbA and PsbB during HL treatment. The fbn5 plants under GL conditions showed almost identical levels of expression of SPS1/2 and HST genes that are involved in PQ-9 biosynthesis (Fig. 8C) (Kim et al. 2015). Under HL stress conditions, the expression of SPS1/2 genes was maximized at the early stages of the stress, a phenomenon which became more remarkable in the fbn5 plants. In contrast, the expression of HST remained unchanged during the HL treatment in both wild-type and fbn5 plants. The expression of HPT, encoding a key enzyme of α-tocopherol biosynthesis, was gradually increased during the HL treatment, but it remained almost constant at low levels in fbn5 plants. These results suggest that FBN5 has implications for α-tocopherol biosynthesis as well as PQ-9 biosynthesis. As shown in Fig. 5A and Table 1, fbn5 plants were hypersensitive to light, resulting in strengthening of photoprotection by dissipation of excess light energy such as via the xanthophyll cycle. The expression of PsbS and the genes involved in the xanthophyll cycle was studied under HL treatment (Fig. 8D). The expression levels of these genes in fbn5 plants were significantly enhanced at all or some of the treatment times examined. This was coincident with the results in Table 2 showing that the amounts of xanthophylls and carotenoids were greater in fbn5 plants than in the wild type under both GL and HL conditions. These results strongly support that the dissipation of excess light energy is accelerated in a proactive manner in fbn5 plants. We further examined the expression of genes involved in ABA signal transduction and jasomonate biosynthesis, which were reported to be functionally related to FBN1a, FBN1b or FBN2 (Fig. 8E, F) (Yang et al. 2006, Youssef et al. 2010). In fbn5 plants, all three genes of jasmonate biosynthesis and NCED of ABA biosynthesis were expressed repressively under both GL and HL conditions. From these results, FBN5 as well as FBN1a/1b/2 is suggested to function in tolerance against environmental stresses directly or indirectly. Discussion The FBN family with a predicted lipocalin motif is a small hydrophobic ligand-binding protein family. Recently, Kim et al. (2015) have reported that FBN5 is required for PQ-9 biosynthesis through its interaction with SPS1/2 and that FBN5-deficient plants were sensitive to cold stress. Some of the FBN family proteins have also been reported to be accumulated under abiotic/biotic stress conditions, and are involved in the response to these stresses as follows: (i) FBN levels (most probably FBN1a and FBN1b) were correlated with protection against photoinhibition under HL, and the ABA response regulators ABI1 and ABI2 associating with FBN1a seemed to control fibrillin expression that was involved in mediating ABA-induced photoprotection (Yang et al. 2006); (ii) FBN1-2 knockdown plants contained less triacylglycerol under high light/cold stress, resulting in a decrease in expression of several jasmonate-induced genes (Youssef et al. 2010); (iii) in the FBN4-knockdown and -knockout plants, the amount of PQ-9 was reported to be drastically decreased, and was susceptible to HL (Singh et al. 2010, Singh et al. 2012). According to the relative protein abundances acquired from the Arabidopsis abundance data sets aggregated in the PaxDb database (Baerenfaller et al. 2012, Wang et al. 2012), FBN5 is one of the minor proteins in the FBN family, as already reported (Lundquist et al. 2012); the protein abundance in leaf of FBN5 was much lower than those of FBN-1a, -1b, -4, -2, -7b, -6 and -3a, and was comparable with those of FBN-10, -8, -7a and -3b. The FBN5 protein abundance in leaf was also comparable with that of SPS1/2 that have been reported to interact physically with FBN5. FBN5 is expressed organ specifically; the protein abundance of FBN5 in individual organs was in the following order of highest to lowest: juvenile leaf > cotyledon > leaf > root > carpel, flower, flower bud, pollen, seed, shoot and silique. Such organ specificity in protein abundance was similar to that in mRNA abundance in this study (Fig. 2B), although the mRNA abundance in flower was considerably higher. The FBN5 protein was located in the stroma, as already reported (Lundquist et al. 2012), which was not affected by HL treatment (Supplementary Fig. S5). While FBN5 is expressed at a low level with respect to protein abundance, the dysfunction of this gene led to critical changes in structural and functional properties of plants as shown by the following evidence revealed by this study. (i) The fbn5 plants grown even under GL conditions at 80 µmol m–2 s–1 showed a drastic decrease in electron transfer activities around PSII, with little change in electron transfer activities around PSI, a phenomenon which was exaggerated under HL stress (Fig. 5; Table 1). (ii) The accumulation of PQ-9 was severely suppressed in fbn5 plants, and both photoactive and non-photoactive PQ pools were almost reduced based on the HPLC analyses and the qP component of NPQ (Fig. 6; Table 1). The content of carotenoids such as xanthophylls, β-carotene and lutein acting as antioxidants, as well as PQ-9, was increased rather than unchanged or decreased (Table 2). These findings were supported by the evidence that there was a remarkable difference in PG osmiophilicity between wild-type and fbn5 plants (Fig. 3G, H, J, K;Supplementary Fig. S6); chloroplasts in fbn5 plants mainly contained electron-transparent (non-osmiophilic, or white) PGs, while wild-type chloroplasts contained many electron-opaque (osmiophilic, or black) PGs. Two types of PGs differing in their osmiophilicity were found by Singh et al. (2010), which is thought to result from the reactivity of osmium oxides with certain biological materials such as prenyl quinones, carotenoids and triacylglycerides. Our findings suggest that PG constituents of antioxidants such as PQ-9, carotenoids and triacylglycerides are drastically reduced, probably reflecting the increased stress sensitivity of the fbn5 plants. Similar results were reported in fbn4-knockdown mutants (Singh et al. 2010). (iii) PAM analyses with fbn5 plants showed that NPQ was much lower than that in wild-type plants although NPQ was increased by the HL treatment; qE, a parameter for short-term control, hardly contributed to the total NPQ; and qI, a parameter for long-term control, was a major contributor of NPQ (Table 1). (iv) Deletion of FBN5 had an opposite influence on the expression of FBN family genes and stress-related genes during the HL treatment (Fig. 8). The fbn5 plants showed the suppression of expression of FBN-1a, -1b and -2 during the HL treatment, as well as the suppression of genes of the PSII reaction center (PsbA and PsbB) and major genes for jasmonate and ABA biosynthesis (LOX2, AOS, AOC2 and NCED3). On the other hand, in the fbn5 plants, the expression of genes involved in PQ-9 biosynthesis (SPS1 and SPS2) and in xanthophyll cycle/biosynthesis (PsbS, ZDS, VDE and ZEP) were considerably enhanced, although their expression patterns were different during HL treatment. (v) The ultrastructure of thylakoids in fbn5 plants was drastically changed: thylakoid membranes were swollen and fragmented, and their grana were unstacked and transformed into loose and disordered structures (Fig. 3H), a phenomenon which was reported with the thylakoid membranes induced by light stress (Chuartzman et al. 2008, Herbstová et al. 2012) and the chloroplasts which were impaired in the reducing sides of PSII (Hagio et al. 2002). Furthermore, in the fbn5 plants, HL treatment caused a marked change in the ultrastructure of chloroplasts in that the grana became more fragmentated (Fig. 3K), although hardly any change in the ultrastructure was observed in the wild-type and fbn5/FBN5 plants (Fig. 3J, L). These structural changes have not been reported with the thylakoid membranes in other fbn mutants such as the fbn1a/fbn1b-knockout mutants of Arabidopsis (Gamez-Arjona et al. 2014), FBN1-2 RNAi (RNA interference) lines of Arabidopsis (Youssef et al. 2010) and FBN4 RNAi lines of apple trees (Singh et al. 2010). The characteristics of fbn5 mutant plants described above can be explained mostly by a decline in the ability to accumulate PQ-9, which causes the limitation of electron transfer from PSII to the Cyt b6f complex to produce ROS (1O2, O2·– and H2O2) by giving excess electrons on the reducing side of PSII to oxygen molecules (Fischer et al. 2013). The redox state of quinone acceptors of PSII are shifted to a reduced state by such excess supply of electrons, which increases the probability of 1O2 and O2·– generation by the charge recombination of P680+ pheophytin– to 3P680 (Rutherford and Krieger-Liszkay 2001) and the reduction of molecular O2 via plastid terminal oxidase (PTOX) (Yu et al. 2014), respectively. Indeed, as shown in Table 1, fbn5 plants have shown greater values of a parameter ΦC which represents the possibility of 1O2 generation by PSII. To protect plants from such photodamage by ROS, plants evolutionarily obtain the mechanisms for both short- and long-term control of the input of light energy to photosynthetic reaction centers (Foyer et al. 2012, Derks et al. 2015, Ruban 2016). In this study, the deletion of FBN5, which represented misconducted the short-term control such as state transitions and xanthophyll cycle which should occur within minutes. Furthermore, FBN5 also did the long-term control such as PSII repair and plant development which should occur on the order of hours through the processes of light-dependent regulation of gene expression. With regard to the relationship between the PQ-9 abundance and phototolerance, Ksas et al. (2015) have clarified that the SPS1-overexpressing lines, which specifically accumulate PQ-9 and its derivative plastochromanol-8, were much more resistant to HL stress than the wild type, showing marked decreases in leaf bleaching, lipid peroxidation and PSII photoinhibition under excess light. Consequently, the fbn5 plants elicited a hypersensitive response in light, which may result from the low yield of PQ-9 and the shift of the reduced form of PQ-9. Our results strongly support the conclusion by Kim et al. (2015) that FBN5 functions as a structural component involved in the biosynthesis of PQ-9 by forming FBN5-B/SPS homodimeric complexes. Besides this function of FBN5, we propose another function in the light stress response. The transcriptional response to increased 1O2 production is clearly different from the response to other ROS such as O2·– and H2O2 (Op den Camp et al. 2003, Dietz et al. 2016). The signal transduction of 1O2 from thylakoids to the cytoplasm/nucleus via the stroma, one type of chloroplast retrograde signaling, forms an integral part of a complex signaling network that is connected to other signaling routes involving phytohormones such as jasmonate (Tikkanen et al. 2014, Dietz et al. 2016, Shumbe et al. 2016). The 1O2 generated mainly by PSII is well known to up-regulate the transcription of jasmonate-responsive genes and genes involved in the biosynthesis of jasmonate. Indeed, in the wild-type plants in this study, the HL treatment markedly enhanced the expression of genes involved in the biosynthesis of jasmonate (LOX2, AOS and AOC2) (Fig. 8F). However, in fbn5 plants, although the production of 1O2 is considered to be elevated under HL and even GL conditions (Table 1), the expression of these genes was down-regulated. A similar phenomenon was reported with Arabidopsis mutants in which FBN-1a, -1b and -2 are all down-regulated (Youssef et al. 2010); FBNs 1a/1b/2 are located in PGs, while FBN5 is found in the stroma. From these results, we speculate that FBN5, as well as FBNs 1a/1b/2, functions as a transmitter of the 1O2 signal pathway in a stromal region. Recently, specific sensors for increased 1O2 have been detected in the thylakoids or via 1O2 reaction products in the stroma; they are oxidation products of carotenoids (such as β-cyclocitral) and lipids (such as the oxylipins 13-hydroxy octadecadienoic acid and/or 13-keto-octadecatrienoic acid, linolenic acid derivatives and precursors of jasmonate) and two chloroplastic proteins, EXECUTER1 (EX1) and EX2 (Wagner et al. 2004, Dietz et al. 2016). FBN5 possesses a lipocalin motif that can bind a small hydrophobic molecule. Furthermore, Malnoë et al. (2018) have reported that the plastid lipocalin, LCNP, is required for sustained photoprotective energy dissipation. Although we were unable to observe a partial translocation of the FBN5 protein from the stroma to thylakoid membranes under the light stress conditions (data not shown), FBN5 is suggested to carry such signaling compounds in the stroma. Further studies will be needed to unveil a functional role for FBN5 in the 1O2 signaling pathway in the chloroplast. Materials and Methods Plant materials and growth conditions All Arabidopsis thaliana (L) Heynh. mutants used in this work were of the Col-0 background. The knockout mutant fbn5 (SALK_064597) was acquired from T-DNA insertion lines of the ABRC. Surface-sterilized wild-type, mutant and transgenic seeds were plated onto Murashige and Skoog (MS) medium with 1% sucrose. The plated seeds were vernalized at 4°C for 3 d in the dark to synchronize germination and then transferred to a growth chamber at 23°C under a 12 h light:12 h dark cycle using GL (photon flux density of 80 µmol m–2 s–1). Two-week-old seedlings were then grown in Jiffy-7 33 mm soil (Jiffy International AS) for 4 weeks under the same conditions as described above. Activation-tagging and freezing-tolerant mutant selection Transformation of calli was performed as described by Otsubo et al. (2007), which was based on the method by Akama et al. (1992). Transformed calli were cooled to −14°C in a freezing bath (NCB-3200, EYELA). The calli were thawed and transferred to shoot-inducing medium (SIM; MS medium supplemented with IAA and trans-zeatin). Surviving transformants were transferred to new SIM plates and ascribed as candidates for freezing-tolerant mutants. RNA extraction, DNA extraction and PCR Total RNA from Arabidopsis leaves was isolated using TRIzol (Invitrogen) according to the manufacturer’s protocol. A 1 μg aliquot of RNA was used as a template for first-strand cDNA synthesis using ReverTra Ace qPCR RT Master Mix with gDNA Remover (Toyobo). Genomic DNA was isolated using the DNeasy Plant Miniprep kit (Qiagen). PCR was performed using EX Taq (TAKARA) or KOD FX (Toyobo). Primer sequences and descriptions are provided in Supplementary Table S1. Complementation of the Arabidopsis fbn5 mutation The full-length FBN5 coding sequences (FBN5-259 and FBN5-273) were amplified from cDNA obtained from Arabidopsis leaves. The amplified products were XbaI/BamHI digested and cloned into the binary vector pCAM35S [the CaMV 35S promoter and the NOS terminator from pBI121 were inserted into the multiple cloning site (MCS) of pCAMBIA1300]. The resulting vectors were then introduced into competent Agrobacterium tumefaciens cells (GV3101) by the freeze–thaw method (Chen et al. 1994). The fbn5 plants were transformed with the Agrobacterium described above, using the floral dip transformation method (Clough and Bent 1998), and selected by their resistance to hygromycin B. Real-time RT–PCR Quantitative RT–PCR (qRT-PCR) was performed using gene-specific primers (Supplementary Table S1) on a LightCycler 480 system using LightCycler 480 SYBR Green I master mix (Roche Diagnostics). The amplification efficiencies of all primers were between 1.8 and 2.2 for all samples tested. We determined the crossing point of each sample using the Second Derivative Maximum method of the software, and calculated relative mRNA levels using the relative standard curve method. The expression of each gene was normalized to the expression of Actin2. Electron microscopy analysis Fully expanded leaves from plants cultured under a 12 h light/12 h dark photoperiod were collected at the indicated times. Small isosceles triangle pieces (base 2 mm and height 5 mm) of leaves were cut with a razor blade and immediately fixed in 1% glutaraldehyde in 20 mM Na-cacodylate buffer, pH 7.4, at 4°C overnight. After washing with Na-cacodylate buffer, the samples were fixed with 1% OsO4 at 4°C for 1.5 h, dehydrated in a graded series of ethanol and then embedded in Epon 812. Ultrathin (70 nm) sections were cut with an Ultracut EM UC7 microtome (Leica) fitted with a diamond knife. Sections were contrasted with 2% aqueous uranyl acetate and lead citrate (Reynolds 1963). Observations were performed using a JEM 1400 Plus transmission electron microscope (Jeol) at 120 kV, equipped with a fully integrated high-resolution camera system (8 million pixel CCD camera). Different sections from at least three different leaf samples were analyzed. Isolation and subfractionation of intact chloroplasts Intact chloroplasts were isolated from 1–2 g of Arabidopsis rosette leaves by the method of Aronsson and Jarvis (2002). To isolate thylakoids and crude stroma from the intact chloroplasts, intact chloroplasts were lysed in a hypertonic medium (50 mM HEPES, pH 8.0, 5 mM MgCl2) for 30 min on ice, and thylakoid membranes were collected by centrifugation at 10,000×g for 10 min. The resultant supernatant, the crude stromal fraction, was further concentrated with an Amicon Ultra-4 10 K centrifugal filter device (Merck). Protein amounts were determined using the Micro BCA protein assay kit (Thermo Fisher Scientific). Immunoblot analysis Proteins were transferred from an SDS–polyacrylamide gel onto a PVDF membrane by electroblotting using a Trans-Blot SD transfer cell (Bio-Rad). Blots were probed with rabbit anti-RbcL (Agrisera) (1:20,000 dilution), anti-PsbA (kindly gifted by Professor Y.Yamamoto, Okayama University) (1:20,000 dilution) or anti-FBN5 (Sigma-Aldrich) (1 µg ml–1) followed by a horseradish peroxidase-conjugated goat anti-rabbit IgG serum (Santa Cruz Biotechnology), and detected using ECL Advance Western Blotting Reagent (GE Healthcare). Chemiluminescence was visualized using a LuminoGraph II (Atto) apparatus running ImageSaver6 software (Atto). Native green gel electrophoresis Separation of different Chl–protein complexes of isolated thylakoid membranes was performed following the method described previously (Allen and Staehelin 1991). The samples of isolated thylakoid membranes were solubilized for 30 min on ice in a buffer solution containing 0.45% (w/v) n-octyl-β-d-glucoside, 0.45% (w/v) n-dodecyl-β-d-maltoside, 0.1% (w/v) lithium dodecyl sulfate and 10% (w/v) sucrose to adjust the ratio of total non-ionic detergents to Chl at 20:1 (w/w). The unsolubilized fragments were removed by centrifugation and the supernatant obtained was resolved on a 7% separating polyacrylamide gel. Measurement of photosynthetic parameters Electron transport activities of PSI and PSII were measured by evolution and consumption, respectively, of oxygen with a Clark-type oxygen electrode at 23°C unless otherwise indicated (Izawa 1980). PSII maximum efficiency and parameters for photochemical and non-photochemical quenching were obtained by use of a Chl fluorometer Mini-PAM (Walz) at 23°C. Saturation pulse Chl fluorescence yield parameters (Fo, Fm, Fs, Fm', Fm'' and Fo'') were recorded as described in the literature (Avenson et al. 2004, Baker et al. 2007, Baker 2008). Before measuring fluorescence emission, the leaves were dark adapted for 10 min. The PSII maximum quantum yield (Fv/Fm) was obtained from Chl a fluorescence as (Fm – Fo)/Fm, where Fo is the initial Chl fluorescence level and Fm is the maximal fluorescence level, determined with a 0.8 s saturation pulse of approximately 5,000 μmol m–2 s–1. Fs is the steady-state fluorescence level under actinic illumination at different photon flux densities (20, 80 and 400 µmol m–2 s–1), while Fm' and Fm'' are the maximum fluorescence in 5 min actinic illumination and in the subsequent 5 min dark recovery, respectively. Fluorescence parameters employed in this study were calculated as follows: qP = (Fm' – Fs)/(Fm' – Fo'), NPQ = (Fm – Fm')/Fm'), qE = Fm/Fm' − Fm/Fm'', qI = Fm/Fm'' – 1 and ΦC = Fs/Fm (Krause and Jahns 2003, Baker et al. 2007, Ahn et al. 2009, Ruban 2016). More than three measurements were made from three different plants for each plant type. Values are represented as means ± SD (n = 3). Lincomycin treatment Detached leaves were harvested from approximately 5-week-old plants, and were put into 20 ml of Tris-buffered saline (TBS) containing 5 mM lincomycin and 0.2% (v/v) Tween-20. Lincomycin was infiltrated into the leaves by a water aspirator for 90 s. Leaf discs treated with lincomycin were immediately placed on wet filter papers and irradiated with two different light intensities (80 and 400 μmol m–2 s–1), prior to PAM measurements. Analysis of pigments, PQ-9 and α-tocopherol For pigment analysis, frozen tissues were ground in liquid nitrogen, and the pigments were extracted in 85% acetone and analyzed with HPLC using an Agilent 1100 HPLC system, basically according to the method of García-Plazaola and Becerril (1999). To analyze PQ-9 and α-tocopherol, the lipids were extracted from frozen leaves by the method of Kruk and Trebst (2008). Supplementary Data Supplementary data are available at PCP online. Funding This work was supported by the Fukuoka Industry, Science & Technology Foundation. Acknowledgments We would like to acknowledge Drs. Yasusi Yamamoto (Okayama University) and Kensuke Kusumi (Kyushu University) for critical reading of the manuscript. We also acknowledge Dr. Shun Hamada (Fukuoka Women’s University) for technical advice on electron microscopy. Disclosures The authors have no conflicts of interest to declare. References Ahn T.K. , Avenson T.J. , Peers G. , Li Z. , Dall’osto L. , Bassi R. ( 2009 ) Investigating energy partitioning during photosynthesis using an expanded quantum yield convention . Chem. Phys . 357 : 151 – 158 . Google Scholar CrossRef Search ADS Akama K. , Shiraishi H. , Ohta S. , Nakamura K. , Okada K. , Shimura Y. ( 1992 ) Efficient transformation of Arabidopsis thaliana: comparison of the efficiencies with various organs, plant ecotypes and Agrobacterium strains . Plant Cell Rep . 12 : 7 – 11 . 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( 2006 ) Protein profiling of plastoglobules in chloroplasts and chromoplasts. A surprising site for differential accumulation of metabolic enzymes . Plant Physiol . 140 : 984 – 997 . Google Scholar CrossRef Search ADS PubMed Yu Q. , Feilke K. , Krieger-Liszkay A. , Beyer P. ( 2014 ) Functional and molecular characterization of plastid terminal oxidase from rice (Oryza sativa) . Biochim. Biophys. Acta 1837 : 1284 – 1292 . Google Scholar CrossRef Search ADS PubMed Abbreviations Abbreviations CaMV Cauliflower mosaic virus FBN fibrillin GL growth light HL high light LHC light-harvesting complex NPQ non-photochemical quenching PG plastoglobule PQ plastoquinone ROS reactive oxygen species RT–PCR reverse transcription–PCR SIM shoot-inducing medium SPP solanesyl diphosphate SPS solanesyl diphosphate synthase © The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. 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Plant and Cell PhysiologyOxford University Press

Published: May 7, 2018

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